UniFrac: a new phylogenetic method for comparing microbial communities

We introduce here a new method for computing differences between microbial communities based on phylogenetic information. This method, UniFrac, measures the phylogenetic distance between sets of taxa in a phylogenetic tree as the fraction of the branch length of the tree that leads to descendants from either one environment or the other, but not both. UniFrac can be used to determine whether communities are significantly different, to compare many communities simultaneously using clustering and ordination techniques, and to measure the relative contributions of different factors, such as chemistry and geography, to similarities between samples. We demonstrate the utility of UniFrac by applying it to published 16S rRNA gene libraries from cultured isolates and environmental clones of bacteria in marine sediment, water, and ice. Our results reveal that (i) cultured isolates from ice, water, and sediment resemble each other and environmental clone sequences from sea ice, but not environmental clone sequences from sediment and water; (ii) the geographical location does not correlate strongly with bacterial community differences in ice and sediment from the Arctic and Antarctic; and (iii) bacterial communities differ between terrestrially impacted seawater (whether polar or temperate) and warm oligotrophic seawater, whereas those in individual seawater samples are not more similar to each other than to those in sediment or ice samples. These results illustrate that UniFrac provides a new way of characterizing microbial communities, using the wealth of environmental rRNA sequences, and allows quantitative insight into the factors that underlie the distribution of lineages among environments.     

Study of bacterial communities in Antarctic coastal waters by a combination of 16S rRNA and 16S rDNA sequencing

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to gamma-Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with beta-Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities.     

Pollution Impacts on Bacterioplankton Diversity in a Tropical Urban Coastal Lagoon System

Despite a great number of published studies addressing estuarine, freshwater and marine bacterial diversity, few have examined urban coastal lagoons in tropical habitats. There is an increasing interest in monitoring opportunistic pathogens as well as indigenous microbial community members in these water bodies by current molecular and microbiological approaches. In this work, bacterial isolates were obtained through selective plate dilution methods to evaluate antibiotic resistances. In addition, 16S rRNA gene libraries were prepared from environmental waters and mixed cultures grown in BHI medium inoculated with Jacarepaguá lagoon waters. Denaturing gradient gel electrophoresis (DGGE) analyses showed distinct community profiles between environmental communities from each studied site and their cultured counterparts. A total of 497 bacterial sequences were analyzed by MOTHUR, yielding 245 operational taxonomic units (OTUs) grouped at 97% similarity. CCA diagrams showcased how several environmental variables affect the distribution of 18 bacterial orders throughout the three distinct habitats. UniFrac metrics and Venn diagrams revealed that bacterial communities retrieved through each experimental approach were significantly different and that only one OTU, closely related to Vibrio cholerae, was shared between them. Potentially pathogenic bacteria were isolated from most sampled environments, fifty percent of which showed antibiotic resistance.     

大亚湾海域可培养细菌鉴定及系统发育分析

对大亚湾海域表层水体的可培养细菌进行分离,测定了分离培养细菌的16S rRNA 基因V3 区序列,并采用该研究中的35 个序列与从GenBank 下载的46 个序列构建了系统树,对细菌进行了分子鉴定。在9 个站点的表层水体中共分离得到70株菌株,分属35 个种。其中α-变形菌纲(Alphaproteobacteria)和放线菌纲(Actinobacteria)为分离菌株最多的两个菌纲,分离率分别为40%和35.7%;而β-变形菌纲(Betaproteobacteria)仅分离到1 株(1.4%)。此外,还分离得到盖球菌属(Kytococcus)、间胞囊菌属(Intrasporangium)等非常见菌株。大亚湾海域细菌的Shannon-Wiener's 多样性指数为3.21。大亚湾海域表层水体可培养细菌丰富的种类多样性以及较高的α-变形菌分离率,说明了该海域具有良好的水质,同时也是潜在的微生物菌种资源库。     

Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated groundwater as revealed by 16S ribosomal DNA analysis

A molecular approach based on the construction of 16S ribosomal DNA clone libraries was used to investigate the microbial diversity of an underground in situ reactor system filled with the original aquifer sediments. After chemical steady state was reached in the monochlorobenzene concentration between the original inflowing groundwater and the reactor outflow, samples from different reactor locations and from inflowing and outflowing groundwater were taken for DNA extraction. Small-subunit rRNA genes were PCR-amplified with primers specific for Bacteria, subsequently cloned and screened for variation by restriction fragment length polymorphism (RFLP). A total of 87 bacterial 16S rDNA genes were sequenced and subjected to phylogenetic analysis. The original groundwater was found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria, by phylotypes not affiliated with currently recognized bacterial phyla, and also by sporulating and non-sporulating sulfate-reducing bacteria. The most occurring clone types obtained from the sediment samples of the reactor were related to the beta-Proteobacteria, dominated by sequences almost identical to the widespread bacterium Alcaligenes faecalis, to low G+C gram-positive bacteria and to Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) within the gamma subclass of Proteobacteria in the upper reactor sector. Although bacterial phylotypes originating from the groundwater outflow of the reactors also grouped within different subdivisions of Proteobacteria and low G+C gram-positive bacteria, most of the 16S rDNA sequences were not associated with the sequence types observed in the reactor samples. Our results suggest that the different environments were inhabited by distinct microbial communities in respect to their taxonomic diversity, particular pronounced between sediment attached microbial communities from the reactor samples and free-living bacteria from the groundwater in- and outflow.     

Analysis of the diversity of aerobic,thermophilic endospore-forming bacteria in two Algerian hot springs using cultural and non-cultural methods

Terrestrial hot environments are important resources for isolation of thermophilic microorganisms. Few studies have been made on microbial diversity of Algerian geothermal sites. This paper reports the diversity of thermophilic, aerobic endospore-forming bacteria from water and sediment samples taken from Hammam Ouled Ali and Hammam Debagh, two hot springs with a wide range of temperatures and a very rich mineral composition, located in the region of Guelma, north-east of Algeria using culture-dependent and culture-independent approaches Sequences of the V4 region of the 16S rRNA gene from environmental DNA extracted from sediment samples were analyzed and a set of isolates from water and sediment have been characterized by phenotypic and molecular methods. Phylogenetic surveys using environmental DNA sequences indicated that three families dominated the two hot springs: Planococcaceae, Bacillaceae, and Paenibacillaceae. Phenotypic characterization revealed the morphological, biochemical, and physiological properties of these microorganisms, all of which exhibited a range of common extracellular enzymatic activities. Amplified ribosomal DNA restriction analysis (ARDRA) was used to cluster isolates into different phylotypic groups and phylogenetic analysis of 16S rRNA gene sequences of selected isolates showed that all were closely related to four genera of thermophilic Bacilli: Bacillus, Anoxybacillus, Geobacillus, and Brevibacillus. Our results provide important insights into the microbial ecology of Guelma hot springs. They showed that the phylogenetic diversity of bacterial communities within the two studied hot springs was mostly aerobic, with the presence of taxonomic groups of great biotechnological interest. Bioprospection of thermozymes and other biomolecules within these communities will probably provide a data basis for their industrial exploitation.     

Prokaryotic diversity of arctic ice shelf microbial mats

The prokaryotic diversity and respiratory activity of microbial mat communities on the Markham Ice Shelf and Ward Hunt Ice Shelf in the Canadian high Arctic were analysed. All heterotrophic isolates and > 95% of bacterial 16S rRNA gene clone library sequences from both ice shelves grouped within the phyla Bacteroidetes , Proteobacteria and Actinobacteria . Clone library analyses showed that the bacterial communities were diverse and varied significantly between the two ice shelves, with the Markham library having a higher estimated diversity (Chao1 = 243; 105 operational taxonomic units observed in 189 clones) than the Ward Hunt library (Chao1 = 106; 52 operational taxonomic units observed in 128 clones). Archaeal 16S rRNA gene clone libraries from both ice shelves were dominated by a single Euryarchaeota sequence, which appears to represent a novel phylotype. Analyses of community activity by radiorespiration assays detected metabolism in mat samples from both ice shelves at temperatures as low as −10°C. These findings provide the first insight into the prokaryotic biodiversity of Arctic ice shelf communities and underscore the importance of these cryo-ecosystems as a rich source of microbiota that are adapted to extreme cold.     

Molecular characterization of the microbial community in hydrogenetic ferromanganese crusts of the Takuyo-Daigo Seamount, northwest Pacific

The abundance and phylogenetic diversity of the microbial community in the hydrogenetic ferromanganese crust, sandy sediment and overlying seawater were investigated using a culture-independent molecular analysis based on the 16S rRNA gene. These samples were carefully collected from the Takuyo-Daigo Seamount, located in the northwest Pacific Ocean, by a remotely operated vehicle. Based on quantitative PCR analysis, Archaea occupy a significant portion of the prokaryotic communities in the ferromanganese crust and the sediment samples, while Bacteria dominated in the seawater samples. Phylotypes belonging to Gammaproteobacteria and to Marine group I (MGI) Crenarchaeota were abundant in clone libraries constructed from the ferromanganese crust and sediment samples, while those belonging to Alphaproteobacteria were abundant in that from the seawater sample. Comparative analysis indicates that over 80% of the total phylotype richness estimates for the crust community were unique as compared with the sediment and seawater communities. Phylotypes related to Nitrosospira belonging to the Betaproteobacteria and those related to Nitrosopumilus belonging to MGI Crenarchaeota were detected in the ferromanganese crust, suggesting that these ammonia-oxidizing chemolithoautotrophs play a role as primary producers in the microbial ecosystem of hydrogenetic ferromanganese crusts that was formed as precipitates from seawater.     

Microbial community composition and function in permanently cold seawater and sediments from an arctic fjord of svalbard

Heterotrophic microbial communities in seawater and sediments metabolize much of the organic carbon produced in the ocean. Although carbon cycling and preservation depend critically on the capabilities of these microbial communities, their compositions and capabilities have seldom been examined simultaneously at the same site. To compare the abilities of seawater and sedimentary microbial communities to initiate organic matter degradation, we measured the extracellular enzymatic hydrolysis rates of 10 substrates (polysaccharides and algal extracts) in surface seawater and bottom water as well as in surface and anoxic sediments of an Arctic fjord. Patterns of enzyme activities differed between seawater and sediments, not just quantitatively, in accordance with higher cell numbers in sediments, but also in their more diversified enzyme spectrum. Sedimentary microbial communities hydrolyzed all of the fluorescently labeled polysaccharide and algal extracts, in most cases at higher rates in subsurface than surface sediments. In seawater, in contrast, only 5 of the 7 polysaccharides and 2 of the 3 algal extracts were hydrolyzed, and hydrolysis rates in surface and deepwater were virtually identical. To compare bacterial communities, 16S rRNA gene clone libraries were constructed from the same seawater and sediment samples; they diverged strongly in composition. Thus, the broader enzymatic capabilities of the sedimentary microbial communities may result from the compositional differences between seawater and sedimentary microbial communities, rather than from gene expression differences among compositionally similar communities. The greater number of phylum- and subphylum-level lineages and operational taxonomic units in sediments than in seawater samples may reflect the necessity of a wider range of enzymatic capabilities and strategies to access organic matter that has already been degraded during passage through the water column. When transformations of marine organic matter are considered, differences in community composition and their different abilities to access organic matter should be taken into account.     

Sulfate-reducing bacteria in tubes constructed by the marine infaunal polychaete Diopatra cuprea

Marine infaunal burrows and tubes greatly enhance solute transport between sediments and the overlying water column and are sites of elevated microbial activity. Biotic and abiotic controls of the compositions and activities of burrow and tube microbial communities are poorly understood. The microbial communities in tubes of the marine infaunal polychaete Diopatria cuprea collected from two different sediment habitats were examined. The bacterial communities in the tubes from a sandy sediment differed from those in the tubes from a muddy sediment. The difference in community structure also extended to the sulfate-reducing bacterial (SRB) assemblage, although it was not as pronounced for this functional group of species. PCR-amplified 16S rRNA gene sequences recovered from Diopatra tube SRB by clonal library construction and screening were all related to the family Desulfobacteriaceae. This finding was supported by phospholipid fatty acid analysis and by hybridization of 16S rRNA probes specific for members of the genera Desulfosarcina, Desulfobacter, Desulfobacterium, Desulfobotulus, Desulfococcus, and Desulfovibrio and some members of the genera Desulfomonas, Desulfuromonas, and Desulfomicrobium with 16S rRNA gene sequences resolved by denaturing gradient gel electrophoresis. Two of six SRB clones from the clone library were not detected in tubes from the sandy sediment. The habitat in which the D. cuprea tubes were constructed had a strong influence on the tube bacterial community as a whole, as well as on the SRB assemblage.     

Bacterial diversity of water and sediment in the Changjiang estuary and coastal area of the East China Sea

The Changjiang estuary and the coastal area of the East China Sea (ECS) represent important interfaces of terrestrial and marine environments. This study included analyses of water and sediments collected during different seasons in these regions to determine the composition of microbial assemblages by means of 16S rRNA gene clone libraries. We retrieved 1946 sequences and 779 distinct operational taxonomic units from 36 clone libraries. Shannon–Weaver diversity index values and rarefaction analysis indicated that bacterial diversity in the sediment samples was much higher than in the water samples. Proteobacteria (72.9%) was the most abundant phylum, followed by Firmicutes (6.4%), Bacteroidetes (4.6%) and Actinobacteria (4.1%). In the water, clone sequences related to Alphaproteobacteria were the most abundant, whereas in the sediment samples, sequences affiliated with Gammaproteobacteria were predominant. Principal coordinate analysis showed that water samples collected from the Changjiang estuary and the ECS clustered separately. However, this spatial pattern could not be observed in sediment samples, which were mainly distinguished from one another by the season. Bacterial diversity in the Changjiang estuary was higher than that in the ECS, which may be the result of the mixing of bacterial communities from the Changjiang River, the estuary and the coastal ocean.     

Differences between betaproteobacterial ammonia-oxidizing communities in marine sediments and those in overlying water

To assess links between betaproteobacterial ammonia-oxidizing bacteria (AOB) in marine sediment and in overlying water, communities in Loch Duich, Scotland, were characterized by analysis of clone libraries and denaturant gradient gel electrophoresis of 16S rRNA gene fragments. Nitrosospira cluster 1-like sequences were isolated from both environments, but different sequence types dominated water and sediment samples. Detailed phylogenetic analysis of marine Nitrosospira cluster 1-like sequences in Loch Duich and surrounding regions suggests the existence of at least two different phylogenetic subgroups, potentially indicative of new lineages within the betaproteobacterial AOB, representing different marine ecotypes.     

Seasonal and spatial diversity of microbial communities in marine sediments of the South China Sea

This study was conducted to characterize the diversity of microbial communities in marine sediments of the South China Sea by means of 16S rRNA gene clone libraries. The results revealed that the sediment samples collected in summer harboured a more diverse microbial community than that collected in winter, Deltaproteobacteria dominated 16S rRNA gene clone libraries from both seasons, followed by Gammaproteobacteria, Acidobacteria, Nitrospirae, Planctomycetes, Firmicutes. Archaea phylotypes were also found. The majority of clone sequences shared greatest similarity to uncultured organisms, mainly from hydrothermal sediments and cold seep sediments. In addition, the sedimentary microbial communities in the coastal sea appears to be much more diverse than that of the open sea. A spatial pattern in the sediment samples was observed that the sediment samples collected from the coastal sea and the open sea clustered separately, a novel microbial community dominated the open sea. The data indicate that changes in environmental conditions are accompanied by significant variations in diversity of microbial communities at the South China Sea.     

Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the northern Gulf of Mexico

Background

Marine sponges can associate with abundant and diverse consortia of microbial symbionts. However, associated bacteria remain unexamined for the majority of host sponges and few studies use phylogenetic metrics to quantify symbiont community diversity. DNA fingerprinting techniques, such as terminal restriction fragment length polymorphisms (T-RFLP), might provide rapid profiling of these communities, but have not been explicitly compared to traditional methods.

Methodology/Principal Findings

We investigated the bacterial communities associated with the marine sponges Hymeniacidon heliophila and Haliclona tubifera, a sympatric tunicate, Didemnum sp., and ambient seawater from the northern Gulf of Mexico by combining replicated clone libraries with T-RFLP analyses of 16S rRNA gene sequences. Clone libraries revealed that bacterial communities associated with the two sponges exhibited lower species richness and lower species diversity than seawater and tunicate assemblages, with differences in species composition among all four source groups. T-RFLP profiles clustered microbial communities by source; individual T-RFs were matched to the majority (80.6%) of clone library sequences, indicating that T-RFLP analysis can be used to rapidly profile these communities. Phylogenetic metrics of community diversity indicated that the two sponge-associated bacterial communities include dominant and host-specific bacterial lineages that are distinct from bacteria recovered from seawater, tunicates, and unrelated sponge hosts. In addition, a large proportion of the symbionts associated with H. heliophila were shared with distant, conspecific host populations in the southwestern Atlantic (Brazil).

Conclusions/Significance

The low diversity and species-specific nature of bacterial communities associated with H. heliophila and H. tubifera represent a distinctly different pattern from other, reportedly universal, sponge-associated bacterial communities. Our replicated sampling strategy, which included samples that reflect the ambient environment, allowed us to differentiate resident symbionts from potentially transient or prey bacteria. Pairing replicated clone library construction with rapid community profiling via T-RFLP analyses will greatly facilitate future studies of sponge-microbe symbioses.     

Coral microbial communities, zooxanthellae and mucus along gradients of seawater depth and coastal pollution

The high incidence of coral disease in shallow coastal marine environments suggests seawater depth and coastal pollution have an impact on the microbial communities inhabiting healthy coral tissues. A study was undertaken to determine how bacterial communities inhabiting tissues of the coral Montastraea annularis change at 5 m, 10 m and 20 m water depth in varying proximity to the urban centre and seaport of Willemstad, Cura?ao, Netherlands Antilles. Analyses of terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA gene sequences show significant differences in bacterial communities of polluted and control localities only at the shallowest seawater depth. Furthermore, distinct differences in bacterial communities were found with increasing water depth. Comparisons of TRFLP peaks with sequenced clone libraries indicate the black band disease cyanobacterium clone CD1C11 is common and most abundant on healthy corals in less than 10 m water depth. Similarly, sequences belonging to a previously unrecognized group of likely phototrophic bacteria, herein referred to as CAB-I, were also more common in shallow water. To assess the influence of environmental and physiologic factors on bacterial community structure, canonical correspondence analysis was performed using explanatory variables associated with: (i) light availability; (ii) seawater pollution; (iii) coral mucus composition; (iv) the community structure of symbiotic algae; and (v) the photosynthetic activity of symbiotic algae. Eleven per cent of the variation in bacterial communities was accounted for by covariation with these variables; the most important being photosynthetically active radiation (sunlight) and the coral uptake of sewage-derived compounds as recorded by the delta(15)N of coral tissue.     

Life in extreme environments: microbial diversity in Great Salt Lake,Utah

Great Salt Lake (GSL) represents one of the world’s most hypersaline environments. In this study, the archaeal and bacterial communities at the North and South arms of the lake were surveyed by cloning and sequencing the 16S rRNA gene. The sampling locations were chosen for high salt concentration and the presence of unique environmental gradients, such as petroleum seeps and high sulfur content. Molecular techniques have not been systematically applied to this extreme environment, and thus the composition and the genetic diversity of microbial communities at GSL remain mostly unknown. This study led to the identification of 58 archaeal and 42 bacterial operational taxonomic units. Our phylogenetic and statistical analyses displayed a high biodiversity of the microbial communities in this environment. In this survey, we also showed that the majority of the 16S rRNA gene sequences within the clone library were distantly related to previously described environmental halophilic archaeal and bacterial taxa and represent novel phylotypes.     

Differences between Betaproteobacterial Ammonia-Oxidizing Communities in Marine Sediments and Those in Overlying Water

To assess links between betaproteobacterial ammonia-oxidizing bacteria (AOB) in marine sediment and in overlying water, communities in Loch Duich, Scotland, were characterized by analysis of clone libraries and denaturant gradient gel electrophoresis of 16S rRNA gene fragments. Nitrosospira cluster 1-like sequences were isolated from both environments, but different sequence types dominated water and sediment samples. Detailed phylogenetic analysis of marine Nitrosospira cluster 1-like sequences in Loch Duich and surrounding regions suggests the existence of at least two different phylogenetic subgroups, potentially indicative of new lineages within the betaproteobacterial AOB, representing different marine ecotypes.     

Molecular characterizations of microbial communities fouling painted and unpainted concrete structures

This study reports the use of culture-independent and culture-dependent approaches to identify naturally occurring communities of Bacteria and Fungi fouling the surfaces of concrete structures with and without an acrylic paint coating in Georgia, USA. Genomic DNA was extracted from four different sites and PCR amplification of bacterial ribosomal RNA (16S rRNA) genes and the internal transcribed spacer (ITS) region of fungal rRNA genes was conducted. Bacterial and fungal community composition was determined by restriction analysis of amplified DNA of eight clone libraries and sequencing. Five bacterial phyla were observed, and representatives of the phylum Cyanobacteria and the classes Betaproteobacteria and Gammaproteobacteria dominated the bacterial clone libraries. The ITS region of rRNA gene sequences revealed the dominant phylotypes in the fungal clone libraries to be most closely related to Alternaria, Cladosporium, Epicoccum and Udeniomyces. The majority of these fungal genera could be cultured from the sites and successfully used to foul concrete in laboratory-based experiments. While the fungal sequences were most closely related to cultured isolates, the vast majority of bacterial sequences in the libraries were related to uncultured environmental clones. Results show phylogenetically distinct microbial populations occurring at the four sites.     

Specific detection, isolation, and characterization of selected, previously uncultured members of the freshwater bacterioplankton community

High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the alpha-Proteobacteria, beta-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental alpha-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.