Metagenomic Analysis of Viral Communities in (Hado)Pelagic Sediments

In this study, we analyzed viral metagenomes (viromes) in the sedimentary habitats of three geographically and geologically distinct (hado)pelagic environments in the northwest Pacific; the Izu-Ogasawara Trench (water depth = 9,760 m) (OG), the Challenger Deep in the Mariana Trench (10,325 m) (MA), and the forearc basin off the Shimokita Peninsula (1,181 m) (SH). Virus abundance ranged from 10⁶ to 10¹¹ viruses/cm³ of sediments (down to 30 cm below the seafloor [cmbsf]). We recovered viral DNA assemblages (viromes) from the (hado)pelagic sediment samples and obtained a total of 37,458, 39,882, and 70,882 sequence reads by 454 GS FLX Titanium pyrosequencing from the virome libraries of the OG, MA, and SH (hado)pelagic sediments, respectively. Only 24−30% of the sequence reads from each virome library exhibited significant similarities to the sequences deposited in the public nr protein database (E-value <10⁻³ in BLAST). Among the sequences identified as potential viral genes based on the BLAST search, 95−99% of the sequence reads in each library were related to genes from single-stranded DNA (ssDNA) viral families, including Microviridae, Circoviridae, and Geminiviridae. A relatively high abundance of sequences related to the genetic markers (major capsid protein [VP1] and replication protein [Rep]) of two ssDNA viral groups were also detected in these libraries, thereby revealing a high genotypic diversity of their viruses (833 genotypes for VP1 and 2,551 genotypes for Rep). A majority of the viral genes predicted from each library were classified into three ssDNA viral protein categories: Rep, VP1, and minor capsid protein. The deep-sea sedimentary viromes were distinct from the viromes obtained from the oceanic and fresh waters and marine eukaryotes, and thus, deep-sea sediments harbor novel viromes, including previously unidentified ssDNA viruses.     

Evaluation of genetic markers from the 16S rRNA gene V2 region for use in quantitative detection of selected Bacteroidales species and human fecal waste by qPCR

Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4 × 10⁴ target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>10³ copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R² ≥ 0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters.     

Host Distributions of Uncultivated Fecal Bacteroidales Bacteria Reveal Genetic Markers for Fecal Source Identification

The purpose of this study was to examine host distribution patterns among fecal bacteria in the order Bacteroidales, with the goal of using endemic sequences as markers for fecal source identification in aquatic environments. We analyzed Bacteroidales 16S rRNA gene sequences from the feces of eight hosts: human, bovine, pig, horse, dog, cat, gull, and elk. Recovered sequences did not match database sequences, indicating high levels of uncultivated diversity. The analysis revealed both endemic and cosmopolitan distributions among the eight hosts. Ruminant, pig, and horse sequences tended to form host- or host group-specific clusters in a phylogenetic tree, while human, dog, cat, and gull sequences clustered together almost exclusively. Many of the human, dog, cat, and gull sequences fell within a large branch containing cultivated species from the genus Bacteroides. Most of the cultivated Bacteroides species had very close matches with multiple hosts and thus may not be useful targets for fecal source identification. A large branch containing cultivated members of the genus Prevotella included cloned sequences that were not closely related to cultivated Prevotella species. Most ruminant sequences formed clusters separate from the branches containing Bacteroides and Prevotella species. Host-specific sequences were identified for pigs and horses and were used to design PCR primers to identify pig and horse sources of fecal pollution in water. The primers successfully amplified fecal DNAs from their target hosts and did not amplify fecal DNAs from other species. Fecal bacteria endemic to the host species may result from evolution in different types of digestive systems.     

Chicken- and duck-associated Bacteroides–Prevotella genetic markers for detecting fecal contamination in environmental water

Bacteroides–Prevotella group is one of the most promising targets for detecting fecal contamination in water environments, principally due to its host-specific distributions and high concentrations in feces of warm-blooded animals. We developed real-time PCR assays for quantifying chicken/duck-, chicken-, and duck-associated Bacteroides–Prevotella 16S rRNA genetic markers (Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac). A reference collection of DNA extracts from 143 individual fecal samples and wastewater treatment plant influent was tested by the newly established markers. The quantification limits of Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac markers in environmental water were 54, 57, and 12 copies/reaction, respectively. It was possible to detect possible fecal contaminations from wild ducks in environmental water with the constructed genetic marker assays, even though the density of total coliforms in the identical water samples was below the detection limit. Chicken/Duck-Bac marker was amplified from feces of wild duck and chicken with the positive ratio of 96 and 61 %, respectively, and no cross-reaction was observed for the other animal feces. Chicken-Bac marker was detected from 70 % of chicken feces, while detected from 39 % of cow feces, 8.3 % of pig feces, and 12 % of swan feces. Duck-Bac marker was detected from 85 % of wild duck feces and cross-reacted with 31 % of cow feces. These levels of detection specificity are common in avian-associated genetic markers previously proposed, which implies that there is a practical limitation in the independent application of avian-associated Bacteroides–Prevotella 16S rRNA genetic markers and a combination with other fecal contamination markers is preferable for detecting fecal contamination in water environments.     

Estimation of the Relative Abundance of Different Bacteroides and Prevotella Ribotypes in Gut Samples by Restriction Enzyme Profiling of PCR-Amplified 16S rRNA Gene Sequences

We describe an approach for determining the genetic composition of Bacteroides and Prevotella populations in gut contents based on selective amplification of 16S rRNA gene sequences (rDNA) followed by cleavage of the amplified material with restriction enzymes. The relative contributions of different ribotypes to total Bacteroides and Prevotella 16S rDNA are estimated after end labelling of one of the PCR primers, and the contribution of Bacteroides and Prevotella sequences to total eubacterial 16S rDNA is estimated by measuring the binding of oligonucleotide probes to amplified DNA. Bacteroides and Prevotella 16S rDNA accounted for between 12 and 62% of total eubacterial 16S rDNA in samples of ruminal contents from six sheep and a cow. Ribotypes 4, 5, 6, and 7, which include most cultivated rumen Prevotella strains, together accounted for between 20 and 86% of the total amplified Bacteroides and Prevotella rDNA in these samples. The most abundant Bacteroides or Prevotella ribotype in four animals, however, was ribotype 8, for which there is only one known cultured isolate, while ribotypes 1 and 2, which include many colonic Bacteroides spp., were the most abundant in two animals. This indicates that some abundant Bacteroides and Prevotella groups in the rumen are underrepresented among cultured rumen Prevotella isolates. The approach described here provides a rapid, convenient, and widely applicable method for comparing the genotypic composition of bacterial populations in gut samples.     

Identification of Nonpoint Sources of Fecal Pollution in Coastal Waters by Using Host-Specific 16S Ribosomal DNA Genetic Markers from Fecal Anaerobes

We describe a new PCR-based method for distinguishing human and cow fecal contamination in coastal waters without culturing indicator organisms, and we show that the method can be used to track bacterial marker sequences in complex environments. We identified two human-specific genetic markers and five cow-specific genetic markers in fecal samples by amplifying 16S ribosomal DNA (rDNA) fragments from members of the genus Bifidobacterium and the Bacteroides-Prevotella group and performing length heterogeneity PCR and terminal restriction fragment length polymorphism analyses. Host-specific patterns suggested that there are species composition differences in the Bifidobacterium and Bacteroides-Prevotella populations of human and cow feces. The patterns were highly reproducible among different hosts belonging to the same species. Additionally, all host-specific genetic markers were detected in water samples collected from areas frequently contaminated with fecal pollution. Ease of detection and longer survival in water made Bacteroides-Prevotella indicators better than Bifidobacterium indicators. Fecal 16S rDNA sequences corresponding to our Bacteroides-Prevotella markers comprised closely related gene clusters, none of which exactly matched previously published Bacteroides or Prevotella sequences. Our method detected host-specific markers in water at pollutant concentrations of 2.8 × 10⁻⁵ to 2.8 × 10⁻⁷ g (dry weight) of feces/liter and 6.8 × 10⁻⁷ g (dry weight) of sewage/liter. Although our aim was to identify nonpoint sources of fecal contamination, the method described here should be widely applicable for monitoring spatial and temporal fluctuations in specific bacterial groups in natural environments.     

Metagenomic analysis of viral community in the Yangtze River expands known eukaryotic and prokaryotic virus diversity in freshwater

Viruses in aquatic ecosystems are characterized by extraordinary abundance and diversity. Thus far, there have been limited studies focused on viral communities in river water systems. Here, we investigated the virome of the Yangtze River Delta using viral metagenomic analysis. The compositions of viral communities from six sampling sites were analyzed and compared. By using library construction and next generation sequencing, contigs and singlet reads similar to viral sequences were classified into 17 viral families, including nine dsDNA viral families, four ssDNA viral families and four RNA viral families. Statistical analysis using Friedman test suggested that there was no significant difference among the six sampling sites (P > 0.05). The viromes in this study were all dominated by the order Caudovirales, and a group of Freshwater phage uvFW species were particularly prevalent among all the samples. The virome from Nanjing presented a unique pattern of viral community composition with a relatively high abundance of family Parvoviridae. Phylogenetic analyses based on virus hallmark genes showed that the Caudovirales order and CRESS-DNA viruses presented high genetic diversity, while viruses in the Microviridae and Parvoviridae families and the Riboviria realm were relatively conservative. Our study provides the first insight into viral community composition in large river ecosystem, revealing the diversity and stability of river water virome, contributing to the proper utilization of freshwater resource.     

Evidence for Extraintestinal Growth of Bacteroidales Originating from Poultry Litter

Water quality monitoring techniques that target microorganisms in the order Bacteroidales are potential alternatives to conventional methods for detection of fecal indicator bacteria. Bacteroidales and members of the genus Bacteroides have been the focus of microbial source tracking (MST) investigations for discriminating sources of fecal pollution (e.g., human or cattle feces) in environmental waters. For accurate source apportionment to occur, one needs to understand both the abundance of Bacteroides in host feces and the survival of these host-associated microbial markers after deposition in the environment. Studies were undertaken to evaluate the abundance, persistence, and potential for growth of Bacteroidales originating from poultry litter under oxic and anoxic environmental conditions. Bacteroidales abundance, as determined by quantitative PCR (qPCR) with GenBac primers and probe, increased 2 to 5 log gene copies ml⁻¹ and 2 log gene copies g litter⁻¹ under most conditions during incubation of poultry litter in a variety of laboratory microcosm and field mesocosm studies. DNA sequencing of the Bacteroidales organisms in the litter identified taxa with sequences corresponding exactly to the GenBac primer and probe sequences and that were closely related to Bacteroides uniformis, B. ovatus, and B. vulgatus. These results suggest that MST studies using qPCR methods targeting Bacteroidales in watersheds that are affected by poultry litter should be interpreted cautiously. Growth of Bacteroidales originating from poultry litter in environmental waters may occur while Bacteroidales growth from other fecal sources declines, thus confounding the interpretation of MST results.     

Metagenomic characterization of airborne viral DNA diversity in the near-surface atmosphere

Airborne viruses are expected to be ubiquitous in the atmosphere but they still remain poorly understood. This study investigated the temporal and spatial dynamics of airborne viruses and their genotypic characteristics in air samples collected from three distinct land use types (a residential district [RD], a forest [FR], and an industrial complex [IC]) and from rainwater samples freshly precipitated at the RD site (RD-rain). Viral abundance exhibited a seasonal fluctuation in the range between 1.7 × 10(6) and 4.0 × 10(7) viruses m(-3), which increased from autumn to winter and decreased toward spring, but no significant spatial differences were observed. Temporal variations in viral abundance were inversely correlated with seasonal changes in temperature and absolute humidity. Metagenomic analysis of air viromes amplified by rolling-circle phi29 polymerase-based random hexamer priming indicated the dominance of plant-associated single-stranded DNA (ssDNA) geminivirus-related viruses, followed by animal-infecting circovirus-related sequences, with low numbers of nanoviruses and microphages-related genomes. Particularly, the majority of the geminivirus-related viruses were closely related to ssDNA mycoviruses that infect plant-pathogenic fungi. Phylogenetic analysis based on the replication initiator protein sequence indicated that the airborne ssDNA viruses were distantly related to known ssDNA viruses, suggesting that a high diversity of viruses were newly discovered. This research is the first to report the seasonality of airborne viruses and their genetic diversity, which enhances our understanding of viral ecology in temperate regions.     

Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton

Virioplankton have a significant role in marine ecosystems, yet we know little of the predominant biological characteristics of aquatic viruses that influence the flow of nutrients and energy through microbial communities. Family A DNA polymerases, critical to DNA replication and repair in prokaryotes, are found in many tailed bacteriophages. The essential role of DNA polymerase in viral replication makes it a useful target for connecting viral diversity with an important biological feature of viruses. Capturing the full diversity of this polymorphic gene by targeted approaches has been difficult; thus, full-length DNA polymerase genes were assembled out of virioplankton shotgun metagenomic sequence libraries (viromes). Within the viromes novel DNA polymerases were common and found in both double-stranded (ds) DNA and single-stranded (ss) DNA libraries. Finding DNA polymerase genes in ssDNA viral libraries was unexpected, as no such genes have been previously reported from ssDNA phage. Surprisingly, the most common virioplankton DNA polymerases were related to a siphovirus infecting an α-proteobacterial symbiont of a marine sponge and not the podoviral T7-like polymerases seen in many other studies. Amino acids predictive of catalytic efficiency and fidelity linked perfectly to the environmental clades, indicating that most DNA polymerase-carrying virioplankton utilize a lower efficiency, higher fidelity enzyme. Comparisons with previously reported, PCR-amplified DNA polymerase sequences indicated that the most common virioplankton metagenomic DNA polymerases formed a new group that included siphoviruses. These data indicate that slower-replicating, lytic or lysogenic phage populations rather than fast-replicating, highly lytic phages may predominate within the virioplankton.     

Distinct Circular Single-Stranded DNA Viruses Exist in Different Soil Types

The potential dependence of virus populations on soil types was examined by electron microscopy, and the total abundance of virus particles in four soil types was similar to that previously observed in soil samples. The four soil types examined differed in the relative abundances of four morphological groups of viruses. Machair, a unique type of coastal soil in western Scotland and Ireland, differed from the others tested in having a higher proportion of tailed bacteriophages. The other soils examined contained predominantly spherical and thin filamentous virus particles, but the Machair soil had a more even distribution of the virus types. As the first step in looking at differences in populations in detail, virus sequences from Machair and brown earth (agricultural pasture) soils were examined by metagenomic sequencing after enriching for circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) virus genomes. Sequences from the family Microviridae (icosahedral viruses mainly infecting bacteria) of CRESS-DNA viruses were predominant in both soils. Phylogenetic analysis of Microviridae major coat protein sequences from the Machair viruses showed that they spanned most of the diversity of the subfamily Gokushovirinae, whose members mainly infect obligate intracellular parasites. The brown earth soil had a higher proportion of sequences that matched the morphologically similar family Circoviridae in BLAST searches. However, analysis of putative replicase proteins that were similar to those of viruses in the Circoviridae showed that they are a novel clade of Circoviridae-related CRESS-DNA viruses distinct from known Circoviridae genera. Different soils have substantially different taxonomic biodiversities even within ssDNA viruses, which may be driven by physicochemical factors.     

Effects of Long Term Antibiotic Therapy on Human Oral and Fecal Viromes

Viruses are integral members of the human microbiome. Many of the viruses comprising the human virome have been identified as bacteriophage, and little is known about how they respond to perturbations within the human ecosystem. The intimate association of phage with their cellular hosts suggests their communities may change in response to shifts in bacterial community membership. Alterations to human bacterial biota can result in human disease including a reduction in the host''s resilience to pathogens. Here we report the ecology of oral and fecal viral communities and their responses to long-term antibiotic therapy in a cohort of human subjects. We found significant differences between the viral communities of each body site with a more heterogeneous fecal virus community compared with viruses in saliva. We measured the relative diversity of viruses, and found that the oral viromes were significantly more diverse than fecal viromes. There were characteristic changes in the membership of oral and fecal bacterial communities in response to antibiotics, but changes in fecal viral communities were less distinguishing. In the oral cavity, an abundance of papillomaviruses found in subjects on antibiotics suggests an association between antibiotics and papillomavirus production. Despite the abundance of papillomaviruses identified, in neither the oral nor the fecal viromes did antibiotic therapy have any significant impact upon overall viral diversity. There was, however, an apparent expansion of the reservoir of genes putatively involved in resistance to numerous classes of antibiotics in fecal viromes that was not paralleled in oral viromes. The emergence of antibiotic resistance in fecal viromes in response to long-term antibiotic therapy in humans suggests that viruses play an important role in the resilience of human microbial communities to antibiotic disturbances.     

Fermentable non-starch polysaccharides increases the abundance of Bacteroides–Prevotella–Porphyromonas in ileal microbial community of growing pigs

Most plant-origin fiber sources used in pig production contains a mixture of soluble and insoluble non-starch polysaccharides (NSP). The knowledge about effects of these sources of NSP on the gut microbiota and its fermentation products is still scarce. The aim of this study was to investigate effects of feeding diets with native sources of NSP on the ileal and fecal microbial composition and the dietary impact on the concentration of short-chain fatty acids (SCFA) and lactic acid. The experiment comprised four diets and four periods in a change-over design with seven post valve t-cecum cannulated growing pigs. The four diets were balanced to be similar in NSP content and included one of four fiber sources, two diets were rich in pectins, through inclusion of chicory forage (CFO) and sugar beet pulp, and two were rich in arabinoxylan, through inclusion of wheat bran (WB) and grass meal. The gut microbial composition was assessed with terminal restriction fragment (TRF) length polymorphism and the abundance of Lactobacillus spp., Enterobacteriaceae, Bacteroides–Prevotella–Porphyromonas and the β-xylosidase gene, xynB, were assessed with quantitative PCR. The gut microbiota did not cluster based on NSP structure (arabinoxylan or pectin) rather, the effect was to a high degree ingredient specific. In pigs fed diet CFO, three TRFs related to Prevotellaceae together consisted of more than 25% of the fecal microbiota, which is about 3 to 23 times higher (P<0.05) than in pigs fed the other diets. Whereas pigs fed diet WB had about 2 to 22 times higher abundance (P<0.05) of Megasphaera elsdenii in feces and about six times higher abundance (P<0.05) of Lactobacillus reuteri in ileal digesta than pigs fed the other diets. The total amount of digested NSP (r=0.57; P=0.002), xylose (r=0.53; P=0.004) and dietary fiber (r=0.60; P=0.001) in ileal digesta were positively correlated with an increased abundance of Bacteroides–Prevotella–Porphyromonas. The effect on SCFA was correlated to specific neutral sugars where xylose increased the ileal butyric acid proportion, whereas arabinose increased the fecal butyric acid proportion. Moreover, chicory pectin increased the acetic acid proportion in both ileal digesta and feces.     

Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r² = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

Stone age diseases and modern AIDS

Background

Recent reports have indicated that single-stranded DNA (ssDNA) viruses in the taxonomic families Geminiviridae, Parvoviridae and Anellovirus may be evolving at rates of ~10^-4 substitutions per site per year (subs/site/year). These evolution rates are similar to those of RNA viruses and are surprisingly high given that ssDNA virus replication involves host DNA polymerases with fidelities approximately 10 000 times greater than those of error-prone viral RNA polymerases. Although high ssDNA virus evolution rates were first suggested in evolution experiments involving the geminivirus maize streak virus (MSV), the evolution rate of this virus has never been accurately measured. Also, questions regarding both the mechanistic basis and adaptive value of high geminivirus mutation rates remain unanswered.

Results

We determined the short-term evolution rate of MSV using full genome analysis of virus populations initiated from cloned genomes. Three wild type viruses and three defective artificial chimaeric viruses were maintained in planta for up to five years and displayed evolution rates of between 7.4 × 10^-4 and 7.9 × 10^-4 subs/site/year.

Conclusion

These MSV evolution rates are within the ranges observed for other ssDNA viruses and RNA viruses. Although no obvious evidence of positive selection was detected, the uneven distribution of mutations within the defective virus genomes suggests that some of the changes may have been adaptive. We also observed inter-strand nucleotide substitution imbalances that are consistent with a recent proposal that high mutation rates in geminiviruses (and possibly ssDNA viruses in general) may be due to mutagenic processes acting specifically on ssDNA molecules.     

Oxygen minimum zones harbour novel viral communities with low diversity

Oxygen minimum zones (OMZs) are oceanographic features that affect ocean productivity and biodiversity, and contribute to ocean nitrogen loss and greenhouse gas emissions. Here we describe the viral communities associated with the Eastern Tropical South Pacific (ETSP) OMZ off Iquique, Chile for the first time through abundance estimates and viral metagenomic analysis. The viral‐to‐microbial ratio (VMR) in the ETSP OMZ fluctuated in the oxycline and declined in the anoxic core to below one on several occasions. The number of viral genotypes (unique genomes as defined by sequence assembly) ranged from 2040 at the surface to 98 in the oxycline, which is the lowest viral diversity recorded to date in the ocean. Within the ETSP OMZ viromes, only 4.95% of genotypes were shared between surface and anoxic core viromes using reciprocal BLASTn sequence comparison. ETSP virome comparison with surface marine viromes (Sargasso Sea, Gulf of Mexico, Kingman Reef, Chesapeake Bay) revealed a dissimilarity of ETSP OMZ viruses to those from other oceanic regions. From the 1.4 million non‐redundant DNA sequences sampled within the altered oxygen conditions of the ETSP OMZ, more than 97.8% were novel. Of the average 3.2% of sequences that showed similarity to the SEED non‐redundant database, phage sequences dominated the surface viromes, eukaryotic virus sequences dominated the oxycline viromes, and phage sequences dominated the anoxic core viromes. The viral community of the ETSP OMZ was characterized by fluctuations in abundance, taxa and diversity across the oxygen gradient. The ecological significance of these changes was difficult to predict; however, it appears that the reduction in oxygen coincides with an increased shedding of eukaryotic viruses in the oxycline, and a shift to unique viral genotypes in the anoxic core.     

Use of a genetically-engineered Escherichia coli strain as a sample process control for quantification of the host-specific bacterial genetic markers

Quantitative PCR (qPCR) assays targeting the host-specific Bacteroides-Prevotella 16S rRNA genetic markers have been proposed as one of the promising approaches to identify the source of fecal contamination in environmental waters. One of the concerns of qPCR assays to environmental samples is the reliability of quantified values, since DNA extraction followed by qPCR assays are usually performed without appropriate sample process control (SPC) and internal amplification controls (IACs). To check the errors in sample processing and improve the reliability of qPCR results, it is essential to evaluate the DNA recovery efficiency and PCR amplification efficiency of the target genetic markers and correct the measurement results. In this study, we constructed a genetically-engineered Escherichia coli K12 strain (designated as strain MG1655 Δlac::kan) as sample process control and evaluated the applicability to environmental water samples. The recovery efficiency of the SPC strain MG1655 Δlac::kan was similar to that of Bacteroides fragilis JCM 11019, when DNA were extracted from water samples spiked with the two bacteria. Furthermore, the SPC was included in the qPCR assays with propidium monoazide (PMA) treatment, which can exclude the genetic markers from dead cells. No significant DNA loss was observed in the PMA treatment. The inclusion of both the SPC (strain MG1655 Δlac::kan) and IAC in qPCR assays with PMA treatment gave the assurance of reliable results of host-specific Bacteroides-Prevotella 16S rRNA genetic markers in environmental water samples.     

Adaptation of the polony technique to quantify Gokushovirinae,a diverse group of single-stranded DNA phage

Advances in metagenomics have revealed the ubiquity of single-stranded DNA (ssDNA) phage belonging to the subfamily Gokushovirinae in the oceans; however, the abundance and ecological roles of this group are unknown. Here, we quantify gokushoviruses through adaptation of the polony method, in which viral template DNA is immobilized in a gel, amplified by PCR, and subsequently detected by hybridization. Primers and probes for this assay were designed based on PCR amplicon diversity of gokushovirus major capsid protein gene sequences from a depth profile in the Gulf of Aqaba, Red Sea sampled in September 2015. At ≥95% identity, these 87 gokushovirus sequences formed 14 discrete clusters with the largest clades showing distinct depth distributions. The application of the polony method enabled the first quantification of gokushoviruses in any environment. The gokushoviruses were most abundant in the upper 40 m of the stratified water column, with a subsurface peak in abundance of 1.26 × 10⁵ viruses ml⁻¹. These findings suggest that discrete gokushovirus genotypes infect bacterial hosts that differentially partition in the water column. Since the designed primers and probe are conserved across marine ecosystems, this polony method can be applied broadly for the quantification of gokushoviruses throughout the global oceans.     

新疆两种土地利用方式下土壤病毒的群落组成与功能特征

病毒作为食物网的重要组成部分,在生态系统中发挥着重要的功能。病毒能够影响宿主的死亡率、群落结构和进化,以及营养元素循环。但由于技术方法的限制,对土壤病毒的群落组成和功能特征还知之甚少。为探索不同土地利用方式下土壤病毒组特征,采集了新疆棉花地和荒漠土壤样品。通过宏病毒组学分析发现,棉花地土壤和荒漠土壤分别注释到20个和15个病毒科,单链DNA（ssDNA）病毒占优势,其中微小噬菌体科（Microviridae）占比最高。仅在棉花地土壤中检测到花椰菜花叶病毒科（Caulimoviridae）、逆转录病毒科（Retroviridae）、裸露病毒科（Nudiviridae）、多分DNA病毒科（Polydnaviridae）、杆状病毒科（Baculoviridae）和囊泡病毒科（Ascoviridae）,其中大部分病毒属于植物病毒和昆虫病毒。本研究推测与土地利用方式相关的人为活动、土壤理化性质以及动植物的差异可能影响土壤病毒的群落组成。通过Virsorter共注释到1824条病毒contigs,主要为微小噬菌体科。进一步利用SEED数据库对病毒功能进行注释,发现两个土壤病毒组注释到的主要功能类似;在SEED level 2水平上,均以"Phage capsid proteins"和"Phage packaging machinery"占比最高。本研究可为进一步探索土壤病毒生态功能和土壤食物网提供数据支持。