Expanding the Known Diversity and Environmental Distribution of an Uncultured Phylogenetic Division of Bacteria

Culture-independent molecular phylogenetic methods were used to explore the breadth of diversity and environmental distribution of members of the division-level “candidate” phylogenetic group WS6, recently discovered in a contaminated aquifer and with no cultivated representatives. A broad diversity of WS6-affiliated sequences were cloned from 7 of 12 environments investigated: mainly from anaerobic sediment environments. The number of sequences representing the WS6 candidate division was increased from 3 to 60 in this study. The extent of phylogenetic divergence (sequence difference) in this candidate division was found to be among the largest of any known bacterial division. This indicates that organisms representing the WS6 phylogenetic division offer a broad diversity of undiscovered biochemical and metabolic novelty. These results provide a framework for the further study of these evidently important kinds of organisms and tools, the sequences, with which to do so.     

New Perspective on Uncultured Bacterial Phylogenetic Division OP11

Organisms belonging to the OP11 candidate phylogenetic division of Bacteria have been detected only in rRNA-based sequence surveys of environmental samples. Preliminary studies indicated that such organisms represented by the sequences are abundant and widespread in nature and highly diverse phylogenetically. In order to document more thoroughly the phylogenetic breadth and environmental distribution of this diverse group of organisms, we conducted further molecular analyses on environmental DNAs. Using PCR techniques and primers directed toward each of the five described subdivisions of OP11, we surveyed 17 environmental DNAs and analyzed rRNA gene sequences in 27 clonal libraries from 14 environments. Ninety-nine new and unique sequences were determined completely, and approximately 200 additional clones were subjected to partial sequencing. Extensive phylogenetic comparisons of the new sequences to those representing other bacterial divisions further resolved the phylogeny of the bacterial candidate division OP11 and identified two new candidate bacterial divisions, OP11-derived 1 (OD1) and Sulphur River 1 (SR1). The widespread environmental distribution of representatives of the bacterial divisions OD1, OP11, and SR1 suggests potentially conspicuous biogeochemical roles for these organisms in their respective environments. The information on environmental distribution offers clues for attempts to culture landmark representatives of these novel bacterial divisions, and the sequences are specific molecular signatures that provide for their identification in other contexts.     

Discovery and characterization of a new bacterial candidate division by an anaerobic sludge digester metagenomic approach

We have constructed a large fosmid library from a mesophilic anaerobic digester and explored its 16S rDNA diversity using a high-density filter DNA–DNA hybridization procedure. We identified a group of 16S rDNA sequences forming a new bacterial lineage named WWE3 (Waste Water of Evry 3). Only one sequence from the public databases shares a sequence identity above 80% with the WWE3 group which hence cannot be affiliated to any known or candidate prokaryotic division. Despite representing a non-negligible fraction (5% of the 16S rDNA sequences) of the bacterial population of this digester, the WWE3 bacteria could not have been retrieved using the conventional 16S rDNA amplification procedure due to their unusual 16S rDNA gene sequence. WWE3 bacteria were detected by polymerase chain reaction (PCR) in various environments (anaerobic digesters, swine lagoon slurries and freshwater biofilms) using newly designed specific PCR primer sets. Fluorescence in situ hybridization (FISH) analysis of sludge samples showed that WWE3 microorganisms are oval-shaped and located deep inside sludge flocs. Detailed phylogenetic analysis showed that WWE3 bacteria form a distinct monophyletic group deeply branching apart from all known bacterial divisions. A new bacterial candidate division status is proposed for this group.     

Novel high-rank phylogenetic lineages within a sulfur spring (Zodletone Spring, Oklahoma), revealed using a combined pyrosequencing-sanger approach

The utilization of high-throughput sequencing technologies in 16S rRNA gene-based diversity surveys has indicated that within most ecosystems, a significant fraction of the community could not be assigned to known microbial phyla. Accurate determination of the phylogenetic affiliation of such sequences is difficult due to the short-read-length output of currently available high-throughput technologies. This fraction could harbor multiple novel phylogenetic lineages that have so far escaped detection. Here we describe our efforts in accurate assessment of the novelty and phylogenetic affiliation of selected unclassified lineages within a pyrosequencing data set generated from source sediments of Zodletone Spring, a sulfide- and sulfur-rich spring in southwestern Oklahoma. Lineage-specific forward primers were designed for 78 putatively novel lineages identified within the pyrosequencing data set, and representative nearly full-length small-subunit (SSU) rRNA gene sequences were obtained by pairing those primers with reverse universal bacterial primers. Of the 78 lineages tested, amplifiable products were obtained for 52, 32 of which had at least one nearly full-length sequence that was representative of the lineage targeted. Analysis of phylogenetic affiliation of the obtained Sanger sequences identified 5 novel candidate phyla and 10 novel candidate classes (within Fibrobacteres, Planctomycetes, and candidate phyla BRC1, GN12, TM6, TM7, LD1, WS2, and GN06) in the data set, in addition to multiple novel orders and families. The discovery of multiple novel phyla within a pilot study of a single ecosystem clearly shows the potential of the approach in identifying novel diversities within the rare biosphere.     

A cryptic, intergeneric cytochrome c oxidase I pseudogene in tyrant flycatchers (family: Tyrannidae)

Nuclear mitochondrial pseudogenes, or "numts", are nonfunctional copies of mitochondrial genes that have been translocated to the nuclear genome. Numts have been used to study differences in mutation rates between the nuclear and mitochondrial genomes, but have also been implicated as troublesome for phylogenetic studies and DNA-based species identification (i.e., DNA barcoding). In this study, a suspected numt discovered during a study of mitochondrial cytochrome c oxidase I (COI) diversity in North American birds was targeted and sequenced from tyrant flycatchers (family: Tyrannidae). In total, the numt was found in five taxa representing two genera. Substitution rates were compared between COI and numt sequences. None of the numt sequences harboured stop codons nor frameshift mutations, but phylogenetic analysis revealed they had accumulated more amino acid substitutions than the mitochondrial COI sequences. Mitochondrial COI appeared to be preferentially amplified in most cases, but methods for numt detection are discussed for cases like this where sequences lack obvious features for identification. Because of its persistence across a broad taxonomic lineage, this numt could form a valuable model system for studying evolution in numts. The full size of the numt and its location within the nuclear genome are yet to be determined.     

The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L.

The diversity of bacteria and archaea associating on the surface and interior of maize roots (Zea mays L.) was investigated. A bacterial 16S rDNA primer was designed to amplify bacterial sequences directly from maize roots by PCR to the exclusion of eukaryotic and chloroplast DNA. The mitochondrial sequence from maize was easily separated from the PCR-amplified bacterial sequences by size fractionation. The culturable component of the bacterial community was also assessed, reflecting a community composition different from that of the clone library. The phylogenetic overlap between organisms obtained by cultivation and those identified by direct PCR amplification of 16S rDNA was 48%. Only 4 bacterial divisions were found in the culture collection, which represented 27 phylotypes, whereas 6 divisions were identified in the clonal analysis, comprising 74 phylotypes, including a member of the OP10 candidate division originally described as a novel division level lineage in a Yellowstone hot spring. The predominant group in the culture collection was the actinobacteria and within the clone library, the a-proteobacteria predominated. The population of maize-associated proteobacteria resembled the proteobacterial population of a typical soil community within which resided a subset of specific plant-associated bacteria, such as Rhizobium- and Herbaspirillum-related phylotypes. The representation of phylotypes within other divisions (OP10 and Acidobacterium) suggests that maize roots support a distinct bacterial community. The diversity within the archaeal domain was low. Of the 50 clones screened, 6 unique sequence types were identified, and 5 of these were highly related to each other (sharing 98% sequence identity). The archaeal sequences clustered with good bootstrap support near Marine group I (crenarchaea) and with Marine group II (euryarchaea) uncultured archaea. The results suggest that maize supports a diverse root-associated microbial community composed of species that for the first time have been described as inhabitants of a plant-root environment.     

Genetic diversity of archaea in deep-sea hydrothermal vent environments.

Molecular phylogenetic analysis of naturally occurring archaeal communities in deep-sea hydrothermal vent environments was carried out by PCR-mediated small subunit rRNA gene (SSU rDNA) sequencing. As determined through partial sequencing of rDNA clones amplified with archaea-specific primers, the archaeal populations in deep-sea hydrothermal vent environments showed a great genetic diversity, and most members of these populations appeared to be uncultivated and unidentified organisms. In the phylogenetic analysis, a number of rDNA sequences obtained from deep-sea hydrothermal vents were placed in deep lineages of the crenarchaeotic phylum prior to the divergence of cultivated thermophilic members of the crenarchaeota or between thermophilic members of the euryarchaeota and members of the methanogen-halophile clade. Whole cell in situ hybridization analysis suggested that some microorganisms of novel phylotypes predicted by molecular phylogenetic analysis were likely present in deep-sea hydrothermal vent environments. These findings expand our view of the genetic diversity of archaea in deep-sea hydrothermal vent environments and of the phylogenetic organization of archaea.     

A global census of nitrogenase diversity

The global diversity of nitrogen-fixing microorganisms was assessed through construction and analysis of an aligned database of 16,989 nifH sequences. We conclude that the diversity of diazotrophs is still poorly described and that many organisms remain to be discovered. Our analyses indicate that diversity is not distributed evenly across phylogenetic groups or across environments and that some of the most diverse assemblages and environments remain the most poorly characterized. The majority of OTUs were rare, falling in the long tail of the frequency distribution. The most dominant OTUs fell into either the Cyanobacteria or the α, β, and γ Proteobacteria, and five of these dominant OTUs do not have any representatives cultivated in isolation. Soils contained the greatest diversity of nifH sequences of all of the environments surveyed. Cluster III, which is dominated by nifH sequences from obligate anaerobes, was found to contain the greatest diversity of all nifH lineages and is also the group for which diversity is the least sampled. Our findings provide context for ongoing efforts to explore diazotroph diversity, indicating specific groups and environments that remain poorly characterized.     

Phototrophic Phylotypes Dominate Mesothermal Microbial Mats Associated with Hot Springs in Yellowstone National Park

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.     

Novel Division Level Bacterial Diversity in a Yellowstone Hot Spring

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609–1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among >300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (≥98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing δ-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.     

Phylogenetic diversity, composition and distribution of bacterioplankton community in the Dongjiang River, China

Bacterioplankton community compositions in the Dongjiang River were characterized using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library construction. Water samples in nine different sites were taken along the mainstem and three tributaries. In total, 24 bands from DGGE gels and 406 clones from the libraries were selected and sequenced, subsequently analyzed for the bacterial diversity and composition of those microbial communities. Bacterial 16S rRNA gene sequences from freshwater bacteria exhibited board phylogenetic diversity, including sequences representing the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteriodetes, Verrucomicrobia, and candidate division TM7. Members of Betaproteobacteria group were the most dominant in all sampling sites, followed by Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria. DGGE profiles and the ∫-LIBSHUFF analysis revealed similar patterns of bacterial diversity among most sampling sites, while spatial distribution variances existed in all sites along the river basin. Statistical analysis showed that bacterial species distribution strongly correlated with environmental variables, such as nitrate and ammonia, suggesting that nitrogen nutrients may shape the microbial community structure and composition in the Dongjiang River. This study had important implications for the comparison with other rivers elsewhere and contributed to the growing data set on the factors that structure bacterial communities in freshwater ecosystems.     

The phylogenetic diversity of metagenomes

Phylogenetic diversity--patterns of phylogenetic relatedness among organisms in ecological communities--provides important insights into the mechanisms underlying community assembly. Studies that measure phylogenetic diversity in microbial communities have primarily been limited to a single marker gene approach, using the small subunit of the rRNA gene (SSU-rRNA) to quantify phylogenetic relationships among microbial taxa. In this study, we present an approach for inferring phylogenetic relationships among microorganisms based on the random metagenomic sequencing of DNA fragments. To overcome challenges caused by the fragmentary nature of metagenomic data, we leveraged fully sequenced bacterial genomes as a scaffold to enable inference of phylogenetic relationships among metagenomic sequences from multiple phylogenetic marker gene families. The resulting metagenomic phylogeny can be used to quantify the phylogenetic diversity of microbial communities based on metagenomic data sets. We applied this method to understand patterns of microbial phylogenetic diversity and community assembly along an oceanic depth gradient, and compared our findings to previous studies of this gradient using SSU-rRNA gene and metagenomic analyses. Bacterial phylogenetic diversity was highest at intermediate depths beneath the ocean surface, whereas taxonomic diversity (diversity measured by binning sequences into taxonomically similar groups) showed no relationship with depth. Phylogenetic diversity estimates based on the SSU-rRNA gene and the multi-gene metagenomic phylogeny were broadly concordant, suggesting that our approach will be applicable to other metagenomic data sets for which corresponding SSU-rRNA gene sequences are unavailable. Our approach opens up the possibility of using metagenomic data to study microbial diversity in a phylogenetic context.     

Multiple lateral transfers of dissimilatory sulfite reductase genes between major lineages of sulfate-reducing prokaryotes

A large fragment of the dissimilatory sulfite reductase genes (dsrAB) was PCR amplified and fully sequenced from 30 reference strains representing all recognized lineages of sulfate-reducing bacteria. In addition, the sequence of the dsrAB gene homologs of the sulfite reducer Desulfitobacterium dehalogenans was determined. In contrast to previous reports, comparative analysis of all available DsrAB sequences produced a tree topology partially inconsistent with the corresponding 16S rRNA phylogeny. For example, the DsrAB sequences of several Desulfotomaculum species (low G+C gram-positive division) and two members of the genus Thermodesulfobacterium (a separate bacterial division) were monophyletic with delta-proteobacterial DsrAB sequences. The most parsimonious interpretation of these data is that dsrAB genes from ancestors of as-yet-unrecognized sulfate reducers within the delta-Proteobacteria were laterally transferred across divisions. A number of insertions and deletions in the DsrAB alignment independently support these inferred lateral acquisitions of dsrAB genes. Evidence for a dsrAB lateral gene transfer event also was found within the delta-Proteobacteria, affecting Desulfobacula toluolica. The root of the dsr tree was inferred to be within the Thermodesulfovibrio lineage by paralogous rooting of the alpha and beta subunits. This rooting suggests that the dsrAB genes in Archaeoglobus species also are the result of an ancient lateral transfer from a bacterial donor. Although these findings complicate the use of dsrAB genes to infer phylogenetic relationships among sulfate reducers in molecular diversity studies, they establish a framework to resolve the origins and diversification of this ancient respiratory lifestyle among organisms mediating a key step in the biogeochemical cycling of sulfur.     

Phylogenetic diversity and ecology of environmental Archaea

On the basis of culture studies, Archaea were thought to be synonymous with extreme environments. However, the large numbers of environmental rRNA gene sequences currently flooding into databases such as GenBank show that these organisms are present in almost all environments examined to date. Large sequence databases and new fast phylogenetic software allow more precise determination of the archaeal phylogenetic tree, but also indicate that our knowledge of archaeal diversity is incomplete. Although it is apparent that Archaea can be found in all environments, the chemistry of their ecological context is mostly unknown.     

Macroscopic biofilms in fracture-dominated sediment that anaerobically oxidize methane

Methane release from seafloor sediments is moderated, in part, by the anaerobic oxidation of methane (AOM) performed by consortia of archaea and bacteria. These consortia occur as isolated cells and aggregates within the sulfate-methane transition (SMT) of diffusion and seep-dominant environments. Here we report on a new SMT setting where the AOM consortium occurs as macroscopic pink to orange biofilms within subseafloor fractures. Biofilm samples recovered from the Indian and northeast Pacific Oceans had a cellular abundance of 10(7) to 10(8) cells cm(-3). This cell density is 2 to 3 orders of magnitude greater than that in the surrounding sediments. Sequencing of bacterial 16S rRNA genes indicated that the bacterial component is dominated by Deltaproteobacteria, candidate division WS3, and Chloroflexi, representing 46%, 15%, and 10% of clones, respectively. In addition, major archaeal taxa found in the biofilm were related to the ANME-1 clade, Thermoplasmatales, and Desulfurococcales, representing 73%, 11%, and 10% of archaeal clones, respectively. The sequences of all major taxa were similar to sequences previously reported from cold seep environments. PhyloChip microarray analysis detected all bacterial phyla identified by the clone library plus an additional 44 phyla. However, sequencing detected more archaea than the PhyloChip within the phyla of Methanosarcinales and Desulfurococcales. The stable carbon isotope composition of the biofilm from the SMT (-35 to -43‰) suggests that the production of the biofilm is associated with AOM. These biofilms are a novel, but apparently widespread, aggregation of cells represented by the ANME-1 clade that occur in methane-rich marine sediments.     

Natural microbial diversity in superficial sediments of Milazzo Harbor (Sicily) and community successions during microcosm enrichment with various hydrocarbons

Hydrocarbon-contaminated superficial sediments collected from the Harbor of Milazzo (Tirrenean Sea, northern Sicily), a zone strongly affected by anthropogenic activities, were examined for in situ biodegradative capacities. A culture-independent molecular phylogenetic approach was used to study the influence of hydrocarbon and nutrient addition on the activity and diversity of the indigenous microbiota during a microcosm evaluation. The autochthonous microbial community in non-polluted sediments was represented by eubacterial phylotypes grouped within Proteobacteria, CFB and Firmicutes. The archaeal domain was represented by members of Marine Group I of Crenarchaeota. The majority of recovered sequences was affiliated with heterotrophic genera Clostridium and Vibrio, typical members of eutrophic coastal environments. Amendments of hydrocarbons and mineral nutrients to microcosms dramatically changed the initial diversity of the microbial community. Only bacterial phylotypes affiliated with Proteobacteria and CFB division were detected. The decrease in diversity observed in several microcosms could be explained by the strong selection for microorganisms belonging to group of marine hydrocarbonoclastic gamma-Proteobacteria, namely Alcanivorax, Cycloclasticus, Marinobacter, Marinobacterium/Neptunomonas and Thalassolituus. This study demonstrated that nutrient amendment to hydrocarbon-contaminated superficial sediments enhanced the indigenous microbial biodegradation activity and that highly specialized marine hydrocarbonoclastic bacteria, representing a minor fraction in the natural microbial community, play an important role in the biodegradation of petroleum hydrocarbons accidentally entering the coastal environment.