Comparative metagenomic and rRNA microbial diversity characterization using archaeal and bacterial synthetic communities

Next‐generation sequencing has dramatically changed the landscape of microbial ecology, large‐scale and in‐depth diversity studies being now widely accessible. However, determining the accuracy of taxonomic and quantitative inferences and comparing results obtained with different approaches are complicated by incongruence of experimental and computational data types and also by lack of knowledge of the true ecological diversity. Here we used highly diverse bacterial and archaeal synthetic communities assembled from pure genomic DNAs to compare inferences from metagenomic and SSU rRNA amplicon sequencing. Both Illumina and 454 metagenomic data outperformed amplicon sequencing in quantifying the community composition, but the outcome was dependent on analysis parameters and platform. New approaches in processing and classifying amplicons can reconstruct the taxonomic composition of the community with high reproducibility within primer sets, but all tested primers sets lead to significant taxon‐specific biases. Controlled synthetic communities assembled to broadly mimic the phylogenetic richness in target environments can provide important validation for fine‐tuning experimental and computational parameters used to characterize natural communities.     

Comparative genomic analysis of hyperthermophilic archaeal Fuselloviridae viruses

The complete genome sequences of two Sulfolobus spindle-shaped viruses (SSVs) from acidic hot springs in Kamchatka (Russia) and Yellowstone National Park (United States) have been determined. These nonlytic temperate viruses were isolated from hyperthermophilic Sulfolobus hosts, and both viruses share the spindle-shaped morphology characteristic of the Fuselloviridae family. These two genomes, in combination with the previously determined SSV1 genome from Japan and the SSV2 genome from Iceland, have allowed us to carry out a phylogenetic comparison of these geographically distributed hyperthermal viruses. Each virus contains a circular double-stranded DNA genome of approximately 15 kbp with approximately 34 open reading frames (ORFs). These Fusellovirus ORFs show little or no similarity to genes in the public databases. In contrast, 18 ORFs are common to all four isolates and may represent the minimal gene set defining this viral group. In general, ORFs on one half of the genome are colinear and highly conserved, while ORFs on the other half are not. One shared ORF among all four genomes is an integrase of the tyrosine recombinase family. All four viral genomes integrate into their host tRNA genes. The specific tRNA gene used for integration varies, and one genome integrates into multiple loci. Several unique ORFs are found in the genome of each isolate.     

Comparative analysis of polychlorinated biphenyl-dechlorinating communities in enrichment cultures using three different molecular screening techniques

The catalysts for many microbially mediated environmental processes such as the dechlorination of polychlorinated biphenyls (PCBs) have been difficult to identify by traditional isolation techniques. Numerous, as yet unsuccessful, attempts have been made to isolate and culture the dechlorinating species. To overcome this limitation, amplified rDNA restriction analysis (ARDRA) of a clone library, denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (TRFLP) were used concurrently to compare their effectiveness for characterizing an enriched microbial community. These methods were applied to enrichment cultures that selectively dechlorinated double-flanked chlorines in the PCB congener 2,3,4,5 chlorinated biphenyl. The methods have different biases, which were apparent from discrepancies in the relative clone frequencies (ARDRA), band intensities (DGGE) or peak heights (TRFLP) from the same enrichment culture. However, each method was effectively qualitative and identified the same organisms: a low G + C Gram-positive eubacterium, an organism most similar to the green non-sulphur bacteria, an Aminobacterium sp. and a Desulfovibrio sp. Overall, in community fingerprinting and preliminary identification, DGGE proved to be the most rapid and effective tool for the monitoring of microorganisms within a highly enriched culture. TRFLP results corroborated DGGE fingerprint analysis; however, identification required the additional step of creating a clone library. ARDRA provided an in-depth analysis of the community and this technique detected slight intraspecies sequence variation in 16S rDNA. These molecular methods are common in environmental microbiology, but rarely are they compared with the same sample site or culture. In general, all three methods detected similar community profiles, but inherent biases resulted in different detection limits for individual OTUs (operational taxonomic units).     

A novel group of diverse Polinton-like viruses discovered by metagenome analysis

Background

The rapidly growing metagenomic databases provide increasing opportunities for computational discovery of new groups of organisms. Identification of new viruses is particularly straightforward given the comparatively small size of viral genomes, although fast evolution of viruses complicates the analysis of novel sequences. Here we report the metagenomic discovery of a distinct group of diverse viruses that are distantly related to the eukaryotic virus-like transposons of the Polinton superfamily.

Results

The sequence of the putative major capsid protein (MCP) of the unusual linear virophage associated with Phaeocystis globosa virus (PgVV) was used as a bait to identify potential related viruses in metagenomic databases. Assembly of the contigs encoding the PgVV MCP homologs followed by comprehensive sequence analysis of the proteins encoded in these contigs resulted in the identification of a large group of Polinton-like viruses (PLV) that resemble Polintons (polintoviruses) and virophages in genome size, and share with them a conserved minimal morphogenetic module that consists of major and minor capsid proteins and the packaging ATPase. With a single exception, the PLV lack the retrovirus-type integrase that is encoded in the genomes of all Polintons and the Mavirus group of virophages. However, some PLV encode a newly identified tyrosine recombinase-integrase that is common in bacteria and bacteriophages and is also found in the Organic Lake virophage group. Although several PLV genomes and individual genes are integrated into algal genomes, it appears likely that most of the PLV are viruses. Given the absence of protease and retrovirus-type integrase, the PLV could resemble the ancestral polintoviruses that evolved from bacterial tectiviruses. Apart from the conserved minimal morphogenetic module, the PLV widely differ in their genome complements but share a gene network with Polintons and virophages, suggestive of multiple gene exchanges within a shared gene pool.

Conclusions

The discovery of PLV substantially expands the emerging class of eukaryotic viruses and transposons that also includes Polintons and virophages. This class of selfish elements is extremely widespread and might have been a hotbed of eukaryotic virus, transposon and plasmid evolution. New families of these elements are expected to be discovered.

     

New insights into the archaeal diversity of a hypersaline microbial mat obtained by a metagenomic approach

A metagenomic approach was carried out in order to study the genetic pool of a hypersaline microbial mat, paying more attention to the archaeal community and, specifically, to the putatively methanogenic members. The main aim of the work was to expand the knowledge of a likely ecologically important archaeal lineage, candidate division MSBL1, which is probably involved in methanogenesis at very high salinities.     

Microbial community structure analysis of a benzoate-degrading halophilic archaeal enrichment

A benzoate-degrading archaeal enrichment was developed using sediment samples from Rozel Point at Great Salt Lake, UT. The enrichment degraded benzoate as the sole carbon source at salinity ranging from 2.0 to 5.0 M NaCl with highest rate of degradation observed at 4.0 M. The enrichment was also tested for its ability to grow on other aromatic compounds such as 4-hydroxybenzoic acid (4-HBA), gentisic acid, protocatechuic acid (PCA), catechol, benzene and toluene as the sole sources of carbon and energy. Of these, the culture only utilized 4-HBA as the carbon source. To determine the initial steps in benzoate degradation pathway, a survey of ring-oxidizing and ring-cleaving genes was performed using degenerate PCR primers. Results showed the presence of 4-hydroxybenzoate 3-monooxygenase (4-HBMO) and protocatechuate 3, 4-dioxygenase (3,4-PCA) genes suggesting that the archaeal enrichment might degrade benzoate to 4-HBA that is further converted to PCA by 4-HBMO and, thus, formed PCA would undergo ring-cleavage by 3,4-PCA to form intermediates that enter the Krebs cycle. Small subunit rRNA gene-based diversity survey revealed that the enrichment consisted entirely of class Halobacteria members belonging to the genera Halopenitus, Halosarcina, Natronomonas, Halosimplex, Halorubrum, Salinarchaeum and Haloterrigena. Of these, Halopenitus was the dominant group accounting for almost 91 % of the total sequences suggesting their potential role in degrading oxygenated aromatic compounds at extreme salinity.     

Identification of novel positive-strand RNA viruses by metagenomic analysis of archaea-dominated Yellowstone hot springs

There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.     

Iron reduction by psychrotrophic enrichment cultures

Psychrotrophic (<20 degrees C) enrichment cultures from deep Pacific marine sediments and Alaskan tundra permafrost reduced ferric iron when using organic acids or H(2) as electron donors. The representative culture W3-7 from the Pacific sediments grew fastest at 10 degrees C, which was 5-fold faster than at 25 degrees C and more than 40-fold faster than at 4 degrees C. Fe(III) reduction was also the fastest at 10 degrees C, which was 2-fold faster than at 25 degrees C and 12-fold faster than at 4 degrees C. Overall, about 80% of the enrichment cultures exhibited microbial Fe(III) reduction under psychrotrophic conditions. These results indicated that microbial iron reduction is likely widespread in cold natural environments and may play important roles in cycling of iron and organic matter over geological times.     

Biodiversity within hot spring microbial mat communities: molecular monitoring of enrichment cultures

We have begun to examine the basis for incongruence between hot spring microbial mat populations detected by cultivation or by 16S rRNA methods. We used denaturing gradient gel electrophoresis (DGGE) to monitor enrichments and isolates plated therefrom. At near extincting inoculum dilutions we observed Chloroflexus-like and cyanobacterial populations whose 16S rRNA sequences have been detected in the New Pit Spring Chloroflexus mat and the Octopus Spring cyanobacterial mat. Cyanobacterial populations enriched from 44 to 54°C and 56 to 63°C samples at near habitat temperatures were similar to those previously detected in mat samples of comparable temperatures. However, a lower temperature enrichment from the higher temperature sample selected for the populations found in the lower temperature sample. Three Thermus populations detected by both DGGE and isolation exemplify even more how enrichment may bias our view of community structure. The most abundant population was adap ted to the habitat temperature (50°C), while populations adapted to 65°C and 70°C were 102- and 104-fold less abundant, respectively. However, enrichment at 70°C favored the least abundant strain. Inoculum dilution and incubation at the habitat temperature favored the more numerically relevant populations. We enriched many other aerobic chemoorganotropic populations at various inoculum dilutions and substrate concentrations, most of whose 16S rRNA sequences have not been detected in mats. A common feature of numerically relevant cyanobacterial, Chloroflexus-like and aerobic chemorganotrophic populations, is that they grow poorly and resist cultivation on solidified medium, suggesting plating bias, and that the medium composition and incubation conditions may not reflect the natural microenvironments these populations inhabit.     

Dynamic viral populations in hypersaline systems as revealed by metagenomic assembly

Viruses of the Bacteria and Archaea play important roles in microbial evolution and ecology, and yet viral dynamics in natural systems remain poorly understood. Here, we created de novo assemblies from 6.4 Gbp of metagenomic sequence from eight community viral concentrate samples, collected from 12 h to 3 years apart from hypersaline Lake Tyrrell (LT), Victoria, Australia. Through extensive manual assembly curation, we reconstructed 7 complete and 28 partial novel genomes of viruses and virus-like entities (VLEs, which could be viruses or plasmids). We tracked these 35 populations across the eight samples and found that they are generally stable on the timescale of days and transient on the timescale of years, with some exceptions. Cross-detection of the 35 LT populations in three previously described haloviral metagenomes was limited to a few genes, and most previously sequenced haloviruses were not detected in our samples, though 3 were detected upon reducing our detection threshold from 90% to 75% nucleotide identity. Similar results were obtained when we applied our methods to haloviral metagenomic data previously reported from San Diego, CA: 10 contigs that we assembled from that system exhibited a variety of detection patterns on a timescale of weeks to 1 month but were generally not detected in LT. Our results suggest that most haloviral populations have a limited or, possibly, a temporally variable global distribution. This study provides high-resolution insight into viral biogeography and dynamics and it places "snapshot" viral metagenomes, collected at a single time and location, in context.     

Molecular analysis of methanogenic archaeal communities in managed and natural upland pasture soils

Grassland management influences soil archaeal communities, which appear to be dominated by nonthermophilic crenarchaeotes. To determine whether methanogenic Archaea associated with the Euryarchaeota lineage are also present in grassland soils, anaerobic microcosms containing both managed (improved) and natural (unimproved) grassland rhizosphere soils were incubated for 28 days to encourage the growth of anaerobic Archaea. The contribution of potential methanogenic organisms to the archaeal community was assessed by the molecular analysis of RNA extracted from soil, using primers targeting all Archaea and Euryarchaeota. Archaeal RT‐PCR products were obtained from all anaerobic microcosms. However, euryarchaeal RT‐PCR products (of putative methanogen origin) were obtained only from anaerobic microcosms of improved soil, their presence coinciding with detectable methane production. Sequence analysis of excised denaturing gradient gel electrophoresis (DGGE) bands revealed the presence of euryarchaeal organisms that could not be detected before anaerobic enrichment. These data indicate that nonmethanogenic Crenarchaeota dominate archaeal communities in grassland soil and suggest that management practices encourage euryarchaeal methanogenic activity.     

Molecular analysis of enrichment cultures of marine ammonia oxidisers

Abstract Marine ammonia oxidising bacteria were enriched by incubation of sea water, amended with ammonium sulphate, and subsequent subculture in liquid inorganic medium. PCR primers were designed to be specific for rDNA sequences from ammonia oxidisers belonging to the β -rsub-group of the proteobacteria. These primers were then used to amplify rRNA genes from ammonia oxidiser enrichment cultures containing heterotrophs. PCR products were recovered from all cultures in which complete ammonia oxidation occurred. Subsequent rDNA sequence analysis indicated the presence of three new lineages within the clade defined by sequences of cultured β -sub-group ammonia oxidisers. Two of the new lineages showed moderate similarity to sequences from pure cultures of ammonia oxidisers previously isolated from marine and brackish environments. The third lineage (AEM-3) was deep branching and occupied an intermediate position between clades defined by Nitrosomonas or Nitrosospira , which were isolated from soil or sewage. The phylogenetic analysis suggests that, in enrichment cultures, the primers are specific for members of the target group, the β -proteobacteria ammonia oxidisers. The results also indicate the presence of previously unknown ammonia oxidisers in marine samples. The approach enabled analysis of ammonia oxidiser enrichments at an early stage and without the requirement for isolation of pure cultures, significantly reducing the time required and facilitating quantitative assessment of relatedness of strains.     

Newly discovered insect RNA viruses in China

Insects are a group of arthropods and the largest group of animals on Earth,with over one million species described to date.Like other life forms,insects suffer from viruses that cause disease and death.Viruses that are pathogenic to beneficial insects cause dramatic economic losses on agriculture.In contrast,viruses that are pathogenic to insect pests can be exploited as attractive biological control agents.All of these factors have led to an explosion in the amount of research into insect viruses in recent years,generating impressive quantities of information on the molecular and cellular biology of these viruses.Due to the wide variety of insect viruses,a better understanding of these viruses will expand our overall knowledge of their virology.Here,we review studies of several newly discovered RNA insect viruses in China.     

Metagenomic analysis of the viral communities in fermented foods

Viruses are recognized as the most abundant biological components on Earth, and they regulate the structure of microbial communities in many environments. In soil and marine environments, microorganism-infecting phages are the most common type of virus. Although several types of bacteriophage have been isolated from fermented foods, little is known about the overall viral assemblages (viromes) of these environments. In this study, metagenomic analyses were performed on the uncultivated viral communities from three fermented foods, fermented shrimp, kimchi, and sauerkraut. Using a high-throughput pyrosequencing technique, a total of 81,831, 70,591 and 69,464 viral sequences were obtained from fermented shrimp, kimchi and sauerkraut, respectively. Moreover, 37 to 50% of these sequences showed no significant hit against sequences in public databases. There were some discrepancies between the prediction of bacteriophages hosts via homology comparison and bacterial distribution, as determined from 16S rRNA gene sequencing. These discrepancies likely reflect the fact that the viral genomes of fermented foods are poorly represented in public databases. Double-stranded DNA viral communities were amplified from fermented foods by using a linker-amplified shotgun library. These communities were dominated by bacteriophages belonging to the viral order Caudovirales (i.e., Myoviridae, Podoviridae, and Siphoviridae). This study indicates that fermented foods contain less complex viral communities than many other environmental habitats, such as seawater, human feces, marine sediment, and soil.     

Environmental shaping of sponge associated archaeal communities

Background

Archaea are ubiquitous symbionts of marine sponges but their ecological roles and the influence of environmental factors on these associations are still poorly understood.

Methodology/Principal Findings

We compared the diversity and composition of archaea associated with seawater and with the sponges Hymeniacidon heliophila, Paraleucilla magna and Petromica citrina in two distinct environments: Guanabara Bay, a highly impacted estuary in Rio de Janeiro, Brazil, and the nearby Cagarras Archipelago. For this we used metagenomic analyses of 16S rRNA and ammonia monooxygenase (amoA) gene libraries. Hymeniacidon heliophila was more abundant inside the bay, while P. magna was more abundant outside and P. citrina was only recorded at the Cagarras Archipelago. Principal Component Analysis plots (PCA) generated using pairwise unweighted UniFrac distances showed that the archaeal community structure of inner bay seawater and sponges was different from that of coastal Cagarras Archipelago. Rarefaction analyses showed that inner bay archaeaoplankton were more diverse than those from the Cagarras Archipelago. Only members of Crenarchaeota were found in sponge libraries, while in seawater both Crenarchaeota and Euryarchaeota were observed. Although most amoA archaeal genes detected in this study seem to be novel, some clones were affiliated to known ammonia oxidizers such as Nitrosopumilus maritimus and Cenarchaeum symbiosum.

Conclusion/Significance

The composition and diversity of archaeal communities associated with pollution-tolerant sponge species can change in a range of few kilometers, probably influenced by eutrophication. The presence of archaeal amoA genes in Porifera suggests that Archaea are involved in the nitrogen cycle within the sponge holobiont, possibly increasing its resistance to anthropogenic impacts. The higher diversity of Crenarchaeota in the polluted area suggests that some marine sponges are able to change the composition of their associated archaeal communities, thereby improving their fitness in impacted environments.     

PHACCS,an online tool for estimating the structure and diversity of uncultured viral communities using metagenomic information

Background  

Phages, viruses that infect prokaryotes, are the most abundant microbes in the world. A major limitation to studying these viruses is the difficulty of cultivating the appropriate prokaryotic hosts. One way around this limitation is to directly clone and sequence shotgun libraries of uncultured viral communities (i.e., metagenomic analyses). PHACCS, Phage Communities from Contig Spectrum, is an online bioinformatic tool to assess the biodiversity of uncultured viral communities. PHACCS uses the contig spectrum from shotgun DNA sequence assemblies to mathematically model the structure of viral communities and make predictions about diversity.     

Degradation of unsaturated hydrocarbons by methanogenic enrichment cultures

Abstract The biodegradability of hydrocarbons under anaerobic conditions was studied in enrichment cultures using mineral media inoculated with sewage sludge or sediment samples of limnic and marine origin. No indication of methanogenic degradation was obtained with either n -hexane, n -hexadecane, n -heptadecane, 1-hexene, cis -2-hexene, trans -2-hexene, isoprene, 1-hexine, benzene, toluene, xylene, cyclohexene, cycloheptatriene, cyclopentadiene, styrene, naphthalene, azulene, or β-carotene. Squalene was incompletely converted to methane and carbon dioxide. Complete degradation was observed with 1-hexadecene. Methanogenic subcultures were maintained on 1-hexadecene and squalene. Both enrichments contained after several transfers Methanospirillum hungatei and Methanothrix soehngenii as prevalent methanogenic bacteria. Acetate (≤80 μ M) was the only intermediary product detected indicating that degradation proceeded via hydrogen-dependent syntrophic β-oxidations. Short rods on hexadecene and cocci on squalene were found to be associated with substrate degradation. The results indicate that terminal double bonds can be sufficient to allow methanogenic degradation of hydrocarbons whereas branching and terminal ring closures may significantly contribute to hydrocarbon stability in anoxic environments.