Rare Phylotypes Research Articles

Rare phylotypes in stone, stool, and urine microbiomes are associated with urinary stone disease.

Introduction: In complex microbial communities, the importance of microbial species at very low abundance levels and their prevalence for overall community structure and function is increasingly being recognized. Clinical microbiome studies on urinary stone disease (USD) have indicated that both the gut and urinary tract microbiota are associated with the onset of the disease and that kidney stones them-selves harbor a complex, yet consistent and viable, microbiome. However, how rare phylotypes contribute to this association remains unclear. Delineating the contribution of rare and common phylotypes to urinary stone disease is important for the development of bacteriotherapies to promote urologic health. Methods: The objectives of the current report were to conduct a metaanalysis of 16S rRNA datasets derived from the kidney stone, stool, and urine samples of participants with or without urinary stone disease. To delineate the impact of rare and common phylotypes, metaanalyses were conducted by first separating rare and common taxa determined by both the frequency and abundance of amplicon sequence variants. Results: Consistent with previous analyses, we found that gut, upper urinary, and lower urinary tract microbiomes were all unique. Rare phylotypes comprised the majority of species observed in all sample types, with kidney stones exhibiting the greatest bias toward rarity, followed by urine and stool. Both rare and common fractions contributed significantly to the differences observed between sample types and health disparity. Furthermore, the rare and common fractions were taxonomically unique across all sample types. A total of 222 and 320 unique rare phylotypes from urine and stool samples were found to be significantly associated with USD. A co-occurrence correlation analysis revealed that rare phylotypes are most important for microbiome structure in stones, followed by urine and stool. Discussion: Collectively, the results indicate that rare phylotypes may be important for the pathophysiology of USD, particularly in the kidney stone matrix, which is inherently a very low microbial biomass niche that can have implications for the diagnosis and treatment of kidney stones. Further studies are needed to investigate the functional significance of rare phylotypes in kidney stone pathogenesis.

Weak environmental adaptation of rare phylotypes sustaining soil multi-element cycles in response to decades-long fertilization

Deciphering the ecological role of soil communities in the maintenance of multiple ecosystem functions is pivotal for the conservation and sustainability of soil biodiversity. However, few studies have investigated niche differentiation of abundant and rare microbiota, as well as their contributions to multiple soil elemental cycles, particularly in agroecosystems that have received decades of intense fertilization. Here, we characterized the environmental thresholds and phylogenetic signals for the environmental adaptation of both abundant and rare microbial subcommunities via amplicon sequencing and metagenomic sequencing and explored their importance in sustaining soil multiple nutrient cycling in agricultural fields that were fertilized for two decades. The results showed that rare taxa exhibited narrower niche breadths and weaker phylogenetic signals than abundant species. The assembly of abundant subcommunity was shaped predominantly by dispersal limitation (explained 71.1 % of the variation in bacteria) and undominated processes (explained 75 % of the variation in fungi), whereas the assembly of rare subcommunity was dominated by homogeneous selection process (explained 100 % of the variation in bacteria and 60 % of the variation in fungi). Soil ammonia nitrogen was the leading factor mediating the balance between stochastic and deterministic processes in both abundant (R2 = 0.15, P < 0.001) and rare (R2 = 0.08, P < 0.001) bacterial communities. Notably, the rare biosphere largely contributed to key soil processes such as carbon (R2bacteria = 0.03, P < 0.05; R2fungi = 0.05, P < 0.05) and nitrogen (R2bacteria = 0.03, P < 0.05; R2fungi = 0.17, P < 0.001) cycling. Collectively, these findings facilitate our understanding of the maintenance of rhizosphere bacterial and fungal diversity in response to agricultural fertilization and highlight the key role of rare taxa in sustaining agricultural ecosystem functions.

Convergent Community Assembly among Globally Separated Acidic Cave Biofilms.

Acidophilic bacteria and archaea inhabit extreme geochemical "islands" that can tell us when and how geographic barriers affect the biogeography of microorganisms. Here, we describe microbial communities from extremely acidic (pH 0 to 1) biofilms, known as snottites, from hydrogen sulfide-rich caves. Given the extreme acidity and subsurface location of these biofilms, and in light of earlier work showing strong geographic patterns among snottite Acidithiobacillus populations, we investigated their structure and diversity in order to understand how geography might impact community assembly. We used 16S rRNA gene cloning and fluorescence in situ hybridization (FISH) to investigate 26 snottite samples from four sulfidic caves in Italy and Mexico. All samples had very low biodiversity and were dominated by sulfur-oxidizing bacteria in the genus Acidithiobacillus. Ferroplasma and other archaea in the Thermoplasmatales ranged from 0 to 50% of total cells, and relatives of the bacterial genera Acidimicrobium and Ferrimicrobium were up to 15% of total cells. Rare phylotypes included Sulfobacillus spp. and members of the phyla "Candidatus Dependentiae" and "Candidatus Saccharibacteria" (formerly TM6 and TM7). Although the same genera of acidophiles occurred in snottites on separate continents, most members of those genera represent substantially divergent populations, with 16S rRNA genes that are only 95 to 98% similar. Our findings are consistent with a model of community assembly where sulfidic caves are stochastically colonized by microorganisms from local sources, which are strongly filtered through environmental selection for extreme acid tolerance, and these different colonization histories are maintained by dispersal restrictions within and among caves. IMPORTANCE Microorganisms that are adapted to extremely acidic conditions, known as extreme acidophiles, are catalysts for rock weathering, metal cycling, and mineral formation in naturally acidic environments. They are also important drivers of large-scale industrial processes such as biomining and contaminant remediation. Understanding the factors that govern their ecology and distribution can help us better predict and utilize their activities in natural and engineered systems. However, extremely acidic habitats are unusual in that they are almost always isolated within circumneutral landscapes. So where did their acid-adapted inhabitants come from, and how do new colonists arrive and become established? In this study, we took advantage of a unique natural experiment in Earth's subsurface to show how isolation may have played a role in the colonization history, community assembly, and diversity of highly acidic microbial biofilms.

Dynamics of actively dividing prokaryotes in the western Mediterranean Sea

Microbial community metabolism and functionality play a key role modulating global biogeochemical processes. However, the metabolic activities and contribution of actively growing prokaryotes to ecosystem energy fluxes remain underexplored. Here we describe the temporal and spatial dynamics of active prokaryotes in the different water masses of the Mediterranean Sea using a combination of bromodeoxyuridine labelling and 16S rRNA gene Illumina sequencing. Bulk and actively dividing prokaryotic communities were drastically different and depth stratified. Alteromonadales were rare in bulk communities (contributing 0.1% on average) but dominated the actively dividing community throughout the overall water column (28% on average). Moreover, temporal variability of actively dividing Alteromonadales oligotypes was evinced. SAR86, Actinomarinales and Rhodobacterales contributed on average 3–3.4% each to the bulk and 11, 8.4 and 8.5% to the actively dividing communities in the epipelagic zone, respectively. SAR11 and Nitrosopumilales contributed less to the actively dividing than to the bulk communities during all the study period. Noticeably, the large contribution of these two taxa to the total prokaryotic communities (23% SAR11 and 26% Nitrosopumilales), especially in the meso- and bathypelagic zones, results in important contributions to actively dividing communities (11% SAR11 and 12% Nitrosopumilales). The intense temporal and spatial variability of actively dividing communities revealed in this study strengthen the view of a highly dynamic deep ocean. Our results suggest that some rare or low abundant phylotypes from surface layers down to the deep sea can disproportionally contribute to the activity of the prokaryotic communities, exhibiting a more dynamic response to environmental changes than other abundant phylotypes, emphasizing the role they might have in community metabolism and biogeochemical processes.

Heterotrophy-coordinated diazotrophy is associated with significant changes of rare taxa in soil microbiome

Soil heterotrophic respiration during decomposition of carbon (C)-rich organic matter plays a vital role in sustaining soil fertility. However, it remains poorly understood whether dinitrogen (N2) fixation occurs in support of soil heterotrophic respiration. In this study, 15N2-tracing indicated that strong N2 fixation occurred during heterotrophic respiration of carbon-rich glucose. Soil organic 15N increased from 0.37 atom% to 2.50 atom% under aerobic conditions and to 4.23 atom% under anaerobic conditions, while the concomitant CO2 flux increased by 12.0-fold under aerobic conditions and 5.18-fold under anaerobic conditions. Soil N2 fixation was completely absent in soils replete with inorganic N, although soil N bioavailability did not alter soil respiration. High-throughput sequencing of the 16S rRNA gene further indicated that: i) under aerobic conditions, only 15.2% of soil microbiome responded positively to glucose addition, and these responses were significantly associated with soil respiration and N2 fixation and ii) under anaerobic conditions, the percentage of responses was even lower at 5.70%. Intriguingly, more than 95% of these responses were originally rare with < 0.5% relative abundance in background soils, including typical N2-fixing heterotrophs such as Azotobacter and Clostridium and well-recognized non-N2-fixing heterotrophs such as Sporosarcina, Agromyces, and Sedimentibacter. These results suggest that only a small portion of the soil microbiome could respond quickly to the amendment of readily accessible organic C in a fluvo-aquic soil and highlighted that rare phylotypes might have played more important roles than previously appreciated in catalyzing soil C and nitrogen turnovers. Our study indicates that N2 fixation could be closely associated with microbial turnover of soil organic C when available in excess.

Prokaryotic community assembly after 40 years of soda solonetz restoration by natural grassland and reclaimed farmland

Solonetz soils are typically characterized with excessive saline-alkali content, and physiochemical methods are often employed for solonetz reclamation. However, increasing lines of evidences have suggested that microbiomes play a pivotal role in sustaining soil fertility than previously appreciated, and it remains poorly understood about the taxonomic identity of microbiomes in solonetz soils and the underlying mechanisms that govern the community assembly of solonetz restoration. In this study, we show that significant increase of rare phylotypes occurred after solonetz restoration by natural grassland and reclaimed farmland, and prokaryotic community assembly was dominated by deterministic rather than stochastic processes. The exchangeable sodium decreased significantly from 27.04% in solonetz, to 4.35% in grassland and 0.52% in farmland. Soil organic carbon (SOC) increased significantly with 4.17- and 2.18- fold in grassland and farmland soils relative to solonetz, respectively. Real-time quantitative PCR indicated drastic increases in the abundance of functional genes responsible for nitrogen fixation and nitrification. High-throughput sequencing of 16S rRNA genes further revealed that 180 and 215 phylotypes could be considered as key taxa showing significant increase after solonetz restoration by grassland and farmland. Intriguingly, more than 96% of these key phylotypes were originally rare phylotypes with <0.3% relative abundance in solonetz, including uncultivable lineages such as Acidobacteria Gp6 and Gp4 and well-recognized phylotypes such as Arthrobacter and Microvirga. The advanced statistical analysis of beta-nearest taxon index indicated that deterministic selection dominated microbial community assembly which was closely associated with high soil organic carbon turnover. These results demonstrated that the conversions of solonetz to grassland and farmland lead to significant changes of soil physicochemical properties which in turn could have likely selected for ecologically and agriculturally important functional microorganisms, and rare phylotypes might play important roles than previously appreciated in reclamation of solonetz.

Absolute quantitation of microbes using 16S rRNA gene metabarcoding: A rapid normalization of relative abundances by quantitative PCR targeting a 16S rRNA gene spike-in standard.

Metabarcoding of the 16S rRNA gene is commonly used to characterize microbial communities, by estimating the relative abundance of microbes. Here, we present a method to retrieve the concentrations of the 16S rRNA gene per gram of any environmental sample using a synthetic standard in minuscule amounts (100 ppm to 1% of the 16S rRNA sequences) that is added to the sample before DNA extraction and quantified by two quantitative polymerase chain reaction (qPCR) reactions. This allows normalizing by the initial microbial density, taking into account the DNA recovery yield. We quantified the internal standard and the total load of 16S rRNA genes by qPCR. The qPCR for the latter uses the exact same primers as those used for Illumina sequencing of the V3‐V4 hypervariable regions of the 16S rRNA gene to increase accuracy. We are able to calculate the absolute concentration of the species per gram of sample, taking into account the DNA recovery yield. This is crucial for an accurate estimate as the yield varied between 40% and 84%. This method avoids sacrificing a high proportion of the sequencing effort to quantify the internal standard. If sacrificing a part of the sequencing effort to the internal standard is acceptable, we however recommend that the internal standard accounts for 30% of the environmental 16S rRNA genes to avoid the PCR bias associated with rare phylotypes. The method proposed here was tested on a feces sample but can be applied more broadly on any environmental sample. This method offers a real improvement of metabarcoding of microbial communities since it makes the method quantitative with limited efforts.

A Comparative Analysis of Microbial DNA Preparation Methods for Use With Massive and Branching Coral Growth Forms.

In the last two decades, over 100 studies have investigated the structure of the coral microbiome. However, as yet there are no standardized methods applied to sample preservation and preparation, with different studies using distinct methods. There have also been several comparisons made of microbiome data generated across different studies, which have not addressed the influence of the methodology employed over each of the microbiome datasets. Here, we assess three different preservation methods; salt saturated dimethyl sulfoxide (DMSO) – EDTA, snap freezing with liquid nitrogen and 4% paraformaldehyde solution, and two different preparation methodologies; bead beating and crushing, that have been applied to study the coral microbiome. We compare the resultant bacterial assemblage data for two coral growth forms, the massive coral Goniastrea edwardsi and the branching coral Isopora palifera. We show that microbiome datasets generated from differing preservation and processing protocols are comparable in composition (presence/absence). Significant discrepancies between preservation and homogenization methods are observed in structure (relative abundance), and in the occurrence and dominance of taxa, with rare (low abundance and low occurrence) phylotypes being the most variable fraction of the microbial community. Finally, we provide evidence to support chemical preservation with DMSO as effective as snap freezing samples for generating reliable and robust microbiome datasets. In conclusion, we recommend where possible a standardized preservation and extraction method be taken up by the field to provide the best possible practices for detailed assessments of symbiotic and conserved bacterial associations.

Characterization of root-associated bacterial community structures in soybean and corn using locked nucleic acid (LNA) oligonucleotide-PCR clamping and 454 pyrosequencing

The community structure and diversity of root-associated bacteria have been tentatively investigated using polymerase chain reaction (PCR) amplification methods in several studies. However, the homology between small submit ribosomal (SSU) rRNA genes of plant plastids and mitochondria and that of bacteria have hindered in these studies. To address this issue, in this paper, we adopted the methods of locked nucleic acid (LNA) oligonucleotide-PCR clamping with 454 pyrosequencing to analysis the root-associated bacterial community compositions in soybean and corn. Results showed that plant chloroplast and mitochondria genes were effectively inhibited from PCR amplification in the root samples with LNA oligonucleotides (LNA (+)), and PCR amplicons with LNA (+) had higher bacterial operational taxonomic unit (OTU) numbers and ACE, Chao1, and Shannon indices, as well as a lower Simpson index than the corresponding samples without LNA oligonucleotides (LNA (–)). Those findings suggested that the methods of this study provide a much more detail description of root-associated bacterial communities. In the soybean LNA (+) sample, Pseudomonas, Bradyrhizobium and Flavobacterium were the three most abundant genera, whereas the top two predominant genera in corn LNA (+) samples were Streptomyces and Niastella. The presence and absence of major genera varied between soybean and corn, suggesting the root-associated bacterial communities differed between two crops. The rare phylotypes and uncultured root-associated bacterial members detected in this study inferred that the root-associated bacterial communities are highly complex and information on their taxonomic affiliates potentially gives the clues for selecting the optimal medium and method to isolate the novel bacteria for further functional analysis.

Normalization of environmental metagenomic DNA enhances the discovery of under-represented microbial community members.

Normalization is a procedure classically employed to detect rare sequences in cellular expression profiles (i.e. cDNA libraries). Here, we present a normalization protocol involving the direct treatment of extracted environmental metagenomic DNA with S1 nuclease, referred to as normalization of metagenomic DNA: NmDNA. We demonstrate that NmDNA, prior to post hoc PCR-based experiments (16S rRNA gene T-RFLP fingerprinting and clone library), increased the diversity of sequences retrieved from environmental microbial communities by detection of rarer sequences. This approach could be used to enhance the resolution of detection of ecologically relevant rare members in environmental microbial assemblages and therefore is promising in enabling a better understanding of ecosystem functioning. This study is the first testing 'normalization' on environmental metagenomic DNA (mDNA). The aim of this procedure was to improve the identification of rare phylotypes in environmental communities. Using hypoliths as model systems, we present evidence that this post-mDNA extraction molecular procedure substantially enhances the detection of less common phylotypes and could even lead to the discovery of novel microbial genotypes within a given environment.

Contrasting Response Patterns of Rice Phyllosphere Bacterial Taxa to Elevated CO2

A vast number of microorganisms colonize the leaf surface of terrestrial plants, known as the phyllosphere, and these microorganisms are thought to be of critical importance in plant growth and health. However, the taxonomic identities and ecological functions of the microorganisms inhabiting the rice phyllosphere remain poorly understood. Using a massive, parallel pyrosequencing technique, we identified the phyllosphere bacterial taxa of four different rice varieties and investigated the microbial response to elevated CO2 (eCO2) in a rice field of a free-air CO2 enrichment (FACE) facility located in Jiangsu Province, China. The results showed that the dominant phylotype, the Enterobacteriaceae family of Gammaproteobacteria, accounted for 70.6%–93.8% of the total bacterial communities in the rice phyllosphere. The dominant phylotype was stimulated by eCO2, with its relative abundance increasing from 70.6%–75.2% at ambient CO2 (aCO2) to 86.5%–93.8% at eCO2 in the phyllosphere of rice varieties IIYou084 (TY-084), YangLiangYou6 (YLY-6), and ZhenXian96 (ZX-96). The rare phylotypes, including the bacterial taxa of Sphingobacteriaceae, Xanthomonadaceae, Oxalobacteraceae, Clostridiaceae, and Pseudomonadaceae, were suppressed and their relative abundance decreased from 13.4%–23.0% at aCO2 to 1.47%–6.11% at eCO2. Furthermore, the bacterial diversity indices decreased at eCO2 in the phyllosphere of the rice varieties TY-084, YLY-6, and ZX-96. In contrast, an opposite response pattern was observed for the rice variety of YangDao8 (YD-8). In the phyllosphere of this variety, the relative abundance of the dominant phylotype, Enterobacteriaceae, decreased from 94.1% at aCO2 to 81.4% at eCO2, while that of the rare phylotypes increased from 3.37% to 6.59%. In addition, eCO2 appeared to stimulate bacterial diversity in the rice variety YD-8. Our results suggest that the phyllosphere microbial response to eCO2 might be relative abundance-dependent in paddy fields.

Only Simpson diversity can be estimated accurately from microbial community fingerprints.

Lalande et al. (Microb. Ecol. 66(3):647-658, 2013) introduced a promising approach to quantify microbial diversity from fingerprinting profiles. Their analysis is based on extrapolating the abundance of the phylotypes detectable in a fingerprint towards the rare phylotypes of the community. By considering a set of reconstructed communities, Lalande et al. obtained a range of estimates for phylotype richness, Shannon diversity and Simpson diversity. They reported narrow ranges indicating accurate estimation, especially for Shannon and Simpson diversities. Here, we show that a much larger set of reconstructed communities than the one considered by Lalande et al. is consistent with the fingerprint. We find that the estimates for phylotype richness and Shannon diversity vary over orders of magnitude, but that the estimates for Simpson diversity are restricted to a narrow range (around 10 %). We conclude that only Simpson diversity can be estimated accurately from fingerprints.

Molecular analysis of point-of-use municipal drinking water microbiology

Little is known about the nature of the microbiology in tap waters delivered to consumers via public drinking water distribution systems (DWDSs). In order to establish a broader understanding of the microbial complexity of public drinking waters we sampled tap water from seventeen different cities between the headwaters of the Arkansas River and the mouth of the Mississippi River and determined the bacterial compositions by pyrosequencing small subunit rRNA genes. Nearly 98% of sequences observed among all systems fell into only 5 phyla: Proteobacteria (35%), Cyanobacteria (29%, including chloroplasts), Actinobacteria (24%, of which 85% were Mycobacterium spp.), Firmicutes (6%), and Bacteroidetes (3.4%). The genus Mycobacterium was the most abundant taxon in the dataset, detected in 56 of 63 samples (16 of 17 cities). Among the more rare phylotypes, considerable variation was observed between systems, and was sometimes associated with the type of source water, the type of disinfectant, or the concentration of the environmental pollutant nitrate. Abundant taxa (excepting Cyanobacteria and chloroplasts) were generally similar from system to system, however, regardless of source water type or local land use. The observed similarity among the abundant taxa between systems may be a consequence of the selective influence of chlorine-based disinfection and the common local environments of DWDS and premise plumbing pipes.

Bacterioplankton community structure in the Arctic waters as revealed by pyrosequencing of 16S rRNA genes

Fjords and open oceans are two typical marine ecosystems in the Arctic region, where glacial meltwater and sea ice meltwater have great effects on the bacterioplankton community structure during the summer season. This study aimed to determine the differences in bacterioplankton communities between these two ecosystems in the Arctic region. We conducted a detailed census of microbial communities in Kongsfjorden (Spitsbergen) and the Chukchi Borderland using high-throughput pyrosequencing of the 16S rRNA gene. Gammaproteobacteria and Bacteroidetes were the dominant members of the bacterioplankton community in Kongsfjorden. By contrast, the most abundant bacterial groups in the surface seawater samples from the Chukchi Borderland were Alphaproteobacteria and Actinobacteria. Differences in bacterial communities were found between the surface and subsurface waters in the investigation area of the Chukchi Borderland, and significant differences in bacterial community structure were also observed in the subsurface water between the shelf and deep basin areas. These results suggest the effect of hydrogeographic conditions on bacterial communities. Ubiquitous phylotypes found in all the investigated samples belonged to a few bacterial groups that dominate marine bacterioplankton communities. The sequence data suggested that changes in environmental conditions result in abundant rare phylotypes and reduced amounts of other phylotypes.

Disturbance induced decoupling between host genetics and composition of the associated microbiome

BackgroundStudies of oyster microbiomes have revealed that a limited number of microbes, including pathogens, can dominate microbial communities in host tissues such as gills and gut. Much of the bacterial diversity however remains underexplored and unexplained, although environmental conditions and host genetics have been implicated. We used 454 next generation 16S rRNA amplicon sequencing of individually tagged PCR reactions to explore the diversity of bacterial communities in gill tissue of the invasive Pacific oyster Crassostrea gigas stemming from genetically differentiated beds under ambient outdoor conditions and after a multifaceted disturbance treatment imposing stress on the host.ResultsWhile the gill associated microbial communities in oysters were dominated by few abundant taxa (i.e. Sphingomonas, Mycoplasma) the distribution of rare bacterial groups correlated to relatedness between the hosts under ambient conditions. Exposing the host to disturbance broke apart this relationship by removing rare phylotypes thereby reducing overall microbial diversity. Shifts in the microbiome composition in response to stress did not result in a net increase in genera known to contain potentially pathogenic strains.ConclusionThe decrease in microbial diversity and the disassociation between population genetic structure of the hosts and their associated microbiome suggest that disturbance (i.e. stress) may play a significant role for the assembly of the natural microbiome. Such community shifts may in turn also feed back on the course of disease and the occurrence of mass mortality events in oyster populations.

A Jungle in There: Bacteria in Belly Buttons are Highly Diverse, but Predictable

The belly button is one of the habitats closest to us, and yet it remains relatively unexplored. We analyzed bacteria and arachaea from the belly buttons of humans from two different populations sampled within a nation-wide citizen science project. We examined bacterial and archaeal phylotypes present and their diversity using multiplex pyrosequencing of 16S rDNA libraries. We then tested the oligarchy hypothesis borrowed from tropical macroecology, namely that the frequency of phylotypes in one sample of humans predicts its frequency in another independent sample. We also tested the predictions that frequent phylotypes (the oligarchs) tend to be common when present, and tend to be more phylogenetically clustered than rare phylotypes. Once rarefied to four hundred reads per sample, bacterial communities from belly buttons proved to be at least as diverse as communities known from other skin studies (on average 67 bacterial phylotypes per belly button). However, the belly button communities were strongly dominated by a few taxa: only 6 phylotypes occurred on >80% humans. While these frequent bacterial phylotypes (the archaea were all rare) are a tiny part of the total diversity of bacteria in human navels (<0.3% of phylotypes), they constitute a major portion of individual reads (∼1/3), and are predictable among independent samples of humans, in terms of both the occurrence and evolutionary relatedness (more closely related than randomly drawn equal sets of phylotypes). Thus, the hypothesis that “oligarchs” dominate diverse assemblages appears to be supported by human-associated bacteria. Although it remains difficult to predict which species of bacteria might be found on a particular human, predicting which species are most frequent (or rare) seems more straightforward, at least for those species living in belly buttons.

Biodiversity of Active and Inactive Bacteria in the Gut Flora of Wood-Feeding Huhu Beetle Larvae (Prionoplus reticularis)

Huhu grubs (Prionoplus reticularis) are wood-feeding beetle larvae endemic to New Zealand and belonging to the family Cerambycidae. Compared to the wood-feeding lower termites, very little is known about the diversity and activity of microorganisms associated with xylophagous cerambycid larvae. To address this, we used pyrosequencing to evaluate the diversity of metabolically active and inactive bacteria in the huhu larval gut. Our estimate, that the gut harbors at least 1,800 phylotypes, is based on 33,420 sequences amplified from genomic DNA and reverse-transcribed RNA. Analysis of genomic DNA- and RNA-derived data sets revealed that 71% of all phylotypes (representing 95% of all sequences) were metabolically active. Rare phylotypes contributed considerably to the richness of the community and were also largely metabolically active, indicating their participation in digestive processes in the gut. The dominant families in the active community (RNA data set) included Acidobacteriaceae (24.3%), Xanthomonadaceae (16.7%), Acetobacteraceae (15.8%), Burkholderiaceae (8.7%), and Enterobacteriaceae (4.1%). The most abundant phylotype comprised 14% of the active community and affiliated with Dyella ginsengisoli (Gammaproteobacteria), suggesting that a Dyella-related organism is a likely symbiont. This study provides new information on the diversity and activity of gut-associated microorganisms that are essential for the digestion of the nutritionally poor diet consumed by wood-feeding larvae. Many huhu gut phylotypes affiliated with insect symbionts or with bacteria present in acidic environments or associated with fungi.

Bacterial community changes during bioremediation of aliphatic hydrocarbon-contaminated soil

The microbial community response during the oxygen biostimulation process of aged oil-polluted soils is poorly documented and there is no reference for the long-term monitoring of the unsaturated zone. To assess the potential effect of air supply on hydrocarbon fate and microbial community structure, two treatments (0 and 0.056 mol h⁻¹ molar flow rate of oxygen) were performed in fixed bed reactors containing oil-polluted soil. Microbial activity was monitored continuously over 2 years throughout the oxygen biostimulation process. Microbial community structure before and after treatment for 12 and 24 months was determined using a dual rRNA/rRNA gene approach, allowing us to characterize bacteria that were presumably metabolically active and therefore responsible for the functionality of the community in this polluted soil. Clone library analysis revealed that the microbial community contained many rare phylotypes. These have never been observed in other studied ecosystems. The bacterial community shifted from Gammaproteobacteria to Actinobacteria during the treatment. Without aeration, the samples were dominated by a phylotype linked to the Streptomyces. Members belonging to eight dominant phylotypes were well adapted to the aeration process. Aeration stimulated an Actinobacteria phylotype that might be involved in restoring the ecosystem studied. Phylogenetic analyses suggested that this phylotype is a novel, deep-branching member of the Actinobacteria related to the well-studied genus Acidimicrobium.

The structure of bacterial communities in the western Arctic Ocean as revealed by pyrosequencing of 16S rRNA genes

Bacterial communities in the surface layer of the oceans consist of a few abundant phylotypes and many rare ones, most with unknown ecological functions and unclear roles in biogeochemical processes. To test hypotheses about relationships between abundant and rare phylotypes, we examined bacterial communities in the western Arctic Ocean using pyrosequence data of the V6 region of the 16S rRNA gene. Samples were collected from various locations in the Chukchi Sea, the Beaufort Sea and Franklin Bay in summer and winter. We found that bacterial communities differed between summer and winter at a few locations, but overall there was no significant difference between the two seasons in spite of large differences in biogeochemical properties. The sequence data suggested that abundant phylotypes remained abundant while rare phylotypes remained rare between the two seasons and among the Arctic regions examined here, arguing against the 'seed bank' hypothesis. Phylotype richness was calculated for various bacterial groups defined by sequence similarity or by phylogeny (phyla and proteobacterial classes). Abundant bacterial groups had higher within-group diversity than rare groups, suggesting that the ecological success of a bacterial lineage depends on diversity rather than on the dominance of a few phylotypes. In these Arctic waters, in spite of dramatic variation in several biogeochemical properties, bacterial community structure was remarkably stable over time and among regions, and any variation was due to the abundant phylotypes rather than rare ones.

Bacterial diversity in a glacier foreland of the high Arctic

Over the past 100 years, Arctic temperatures have increased at almost twice the global average rate. One consequence is the acceleration of glacier retreat, exposing new habitats that are colonized by microorganisms whose diversity and function are unknown. Here, we characterized bacterial diversity along two approximately parallel chronosequences in an Arctic glacier forefield that span six time points following glacier retreat. We assessed changes in phylotype richness, evenness and turnover rate through the analysis of 16S rRNA gene sequences recovered from 52 samples taken from surface layers along the chronosequences. An average of 4500 sequences was obtained from each sample by 454 pyrosequencing. Using parametric methods, it was estimated that bacterial phylotype richness was high, and that it increased significantly from an average of 4000 (at a threshold of 97% sequence similarity) at locations exposed for 5 years to an average of 7050 phylotypes per 0.5 g of soil at sites that had been exposed for 150 years. Phylotype evenness also increased over time, with an evenness of 0.74 for 150 years since glacier retreat reflecting large proportions of rare phylotypes. The bacterial species turnover rate was especially high between sites exposed for 5 and 19 years. The level of bacterial diversity present in this High Arctic glacier foreland was comparable with that found in temperate and tropical soils, raising the question whether global patterns of bacterial species diversity parallel that of plants and animals, which have been found to form a latitudinal gradient and be lower in polar regions compared with the tropics.