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As the atmospheric CO(2) concentration rises, more CO(2) will dissolve in the oceans, leading to a reduction in pH. Effects of ocean acidification on bacterial communities have mainly been studied in biologically complex systems, in which indirect effects, mediated through food web interactions, come into play. These approaches come close to nature but suffer from low replication and neglect seasonality. To comprehensively investigate direct pH effects, we conducted highly-replicated laboratory acidification experiments with the natural bacterial community from Helgoland Roads (North Sea). Seasonal variability was accounted for by repeating the experiment four times (spring, summer, autumn, winter). Three dilution approaches were used to select for different ecological strategies, i.e. fast-growing or low-nutrient adapted bacteria. The pH levels investigated were in situ seawater pH (8.15-8.22), pH 7.82 and pH 7.67, representing the present-day situation and two acidification scenarios projected for the North Sea for the year 2100. In all seasons, both automated ribosomal intergenic spacer analysis and 16S ribosomal amplicon pyrosequencing revealed pH-dependent community shifts for two of the dilution approaches. Bacteria susceptible to changes in pH were different members of Gammaproteobacteria, Flavobacteriaceae, Rhodobacteraceae, Campylobacteraceae and further less abundant groups. Their specific response to reduced pH was often context-dependent. Bacterial abundance was not influenced by pH. Our findings suggest that already moderate changes in pH have the potential to cause compositional shifts, depending on the community assembly and environmental factors. By identifying pH-susceptible groups, this study provides insights for more directed, in-depth community analyses in large-scale and long-term experiments.

In Eastern Boundary Upwelling Systems nutrient-rich waters are transported to the ocean surface, fuelling high photoautotrophic primary production. Subsequent heterotrophic decomposition of the produced biomass increases the oxygen-depletion at intermediate water depths, which can result in the formation of oxygen minimum zones (OMZ). OMZs can sporadically accumulate hydrogen sulfide (H2S), which is toxic to most multicellular organisms and has been implicated in massive fish kills. During a cruise to the OMZ off Peru in January 2009 we found a sulfidic plume in continental shelf waters, covering an area >5500 km(2), which contained ∼2.2×10(4) tons of H2S. This was the first time that H2S was measured in the Peruvian OMZ and with ∼440 km(3) the largest plume ever reported for oceanic waters. We assessed the phylogenetic and functional diversity of the inhabiting microbial community by high-throughput sequencing of DNA and RNA, while its metabolic activity was determined with rate measurements of carbon fixation and nitrogen transformation processes. The waters were dominated by several distinct γ-, δ- and ε-proteobacterial taxa associated with either sulfur oxidation or sulfate reduction. Our results suggest that these chemolithoautotrophic bacteria utilized several oxidants (oxygen, nitrate, nitrite, nitric oxide and nitrous oxide) to detoxify the sulfidic waters well below the oxic surface. The chemolithoautotrophic activity at our sampling site led to high rates of dark carbon fixation. Assuming that these chemolithoautotrophic rates were maintained throughout the sulfidic waters, they could be representing as much as ∼30% of the photoautotrophic carbon fixation. Postulated changes such as eutrophication and global warming, which lead to an expansion and intensification of OMZs, might also increase the frequency of sulfidic waters. We suggest that the chemolithoautotrophically fixed carbon may be involved in a negative feedback loop that could fuel further sulfate reduction and potentially stabilize the sulfidic OMZ waters.

The global widespread use of antimicrobials and accompanying increase in resistant bacterial strains is of major public health concern. Wastewater systems and wastewater treatment plants are considered a niche for antibiotic resistance genes (ARGs), with diverse microbial communities facilitating ARG transfer via mobile genetic element (MGE). In contrast to hospital sewage, wastewater from other health care facilities is still poorly investigated. At the instance of a nursing home located in south-west Germany, in the present study, shotgun metagenomics was used to investigate the impact on wastewater of samples collected up- and down-stream in different seasons. Microbial composition, ARGs and MGEs were analyzed using different annotation approaches with various databases, including Antibiotic Resistance Ontologies (ARO), integrons and plasmids. Our analysis identified seasonal differences in microbial communities and abundance of ARG and MGE between samples from different seasons. However, no obvious differences were detected between up- and downstream samples. The results suggest that, in contrast to hospitals, sewage from the nursing home does not have a major impact on ARG or MGE in wastewater, presumably due to much less intense antimicrobial usage. Possible limitations of metagenomic studies using high-throughput sequencing for detection of genes that seemingly confer antibiotic resistance are discussed.

Herpes simplex virus 1 (HSV-1) is an important human pathogen and a paradigm for virus-induced host shut-off. Here we show that global changes in transcription and RNA processing and their impact on translation can be analysed in a single experimental setting by applying 4sU-tagging of newly transcribed RNA and ribosome profiling to lytic HSV-1 infection. Unexpectedly, we find that HSV-1 triggers the disruption of transcription termination of cellular, but not viral, genes. This results in extensive transcription for tens of thousands of nucleotides beyond poly(A) sites and into downstream genes, leading to novel intergenic splicing between exons of neighbouring cellular genes. As a consequence, hundreds of cellular genes seem to be transcriptionally induced but are not translated. In contrast to previous reports, we show that HSV-1 does not inhibit co-transcriptional splicing. Our approach thus substantially advances our understanding of HSV-1 biology and establishes HSV-1 as a model system for studying transcription termination. Herpes simplex virus 1 (HSV-1) efficiently shuts down host gene expression in infected cells. Here Rutkowski et al . analyse the genome-wide changes in transcription and translation in infected cells, and show that HSV-1 triggers an extensive disruption of transcription termination of cellular genes.

Bathymodiolus mussels live in symbiosis with intracellular sulfur-oxidizing (SOX) bacteria that provide them with nutrition. We sequenced the SOX symbiont genomes from two Bathymodiolus species. Comparison of these symbiont genomes with those of their closest relatives revealed that the symbionts have undergone genome rearrangements, and up to 35% of their genes may have been acquired by horizontal gene transfer. Many of the genes specific to the symbionts were homologs of virulence genes. We discovered an abundant and diverse array of genes similar to insecticidal toxins of nematode and aphid symbionts, and toxins of pathogens such as Yersinia and Vibrio. Transcriptomics and proteomics revealed that the SOX symbionts express the toxin-related genes (TRGs) in their hosts. We hypothesize that the symbionts use these TRGs in beneficial interactions with their host, including protection against parasites. This would explain why a mutualistic symbiont would contain such a remarkable ‘arsenal’ of TRGs. Although bacteria are commonly associated with causing illness, many are actually beneficial to the organism they live in or on. The phenomenon of one species helping another to survive is known as symbiosis. Animals thrive at hydrothermal vents in the deep sea because of their partnerships with symbiotic bacteria. The bacteria use the geochemical energy found at hydrothermal vents to convert carbon into sugars, thus providing their animal hosts with essential nutrients. Unlike the symbiotic communities that associate with humans and other mammals, in which thousands of bacterial species co-exist, deep-sea mussels associate with just one or two species of symbiotic bacteria. This relative simplicity is ideal for investigating how the intimate associations between animals and bacteria work. Genes contain the instructions cells and organisms need to survive, and so one way that researchers investigate symbiosis is by studying the genes of the organisms involved. Such studies of beneficial bacteria are beginning to reveal that the molecular mechanisms involved in symbiosis are remarkably similar to those responsible for the harmful effects produced by some bacteria. By performing genetic sequencing on the symbiotic bacteria from deep-sea mussels, Sayavedra et al. have discovered that the bacteria have an unusually large number of toxin-like genes, and that all of these genes are active in the bacteria when they are inside host mussels. This was unexpected, as the bacteria are known to benefit their mussel hosts. The toxin-like genes from the symbiotic bacteria are similar to toxins found in the bacteria that cause diseases such as cholera and the plague in humans and other animals. Sayavedra et al. suggest that the symbiotic bacteria have ‘tamed’ these toxins to use them in beneficial interactions with their host. For example, some of the toxins could help the bacteria and mussels to recognize and interact with each other, and others could help to protect the mussel host from its natural enemies. The next step will be to test these ideas, which will be challenging as the mussels cannot be bred in the laboratory.

Parentally biased expression of transcripts (genomic imprinting) in adult tissues, including the brain, can influence and possibly drive the evolution of behavioral traits. We have previously found that paternally determined cues are involved in population-specific mate choice decisions between two populations of the Western house mouse (Mus musculus domesticus). Here, we ask whether this could be mediated by genomically imprinted transcripts that are subject to fast differentiation between these populations. We focus on three organs that are of special relevance for mate choice and behavior: The vomeronasal organ (VNO), the hypothalamus, and the liver. To first identify candidate transcripts at a genome-wide scale, we used reciprocal crosses between M. m. domesticus and M. m. musculus inbred strains and RNA sequencing of the respective tissues. Using a false discovery cutoff derived from mock reciprocal cross comparisons, we find a total of 66 imprinted transcripts, 13 of which have previously not been described as imprinted. The largest number of imprinted transcripts were found in the hypothalamus; fewer were found in the VNO, and the least were found in the liver. To assess molecular differentiation and imprinting in the wild-derived M. m. domesticus populations, we sequenced the RNA of the hypothalamus from individuals of these populations. This confirmed the presence of the above identified transcripts also in wild populations and allowed us to search for those that show a high genetic differentiation between these populations. Our results identify the Ube3a–Snrpn imprinted region on chromosome 7 as a region that encompasses the largest number of previously not described transcripts with paternal expression bias, several of which are at the same time highly differentiated. For four of these, we confirmed their imprinting status via single nucleotide polymorphism-specific pyrosequencing assays with RNA from reciprocal crosses. In addition, we find the paternally expressed Peg13 transcript within the Trappc9 gene region on chromosome 15 to be highly differentiated. Interestingly, both regions have been implicated in Prader–Willi nervous system disorder phenotypes in humans. We suggest that these genomically imprinted regions are candidates for influencing the population-specific mate-choice in mice.

Residual microorganisms and/or re-infections are a major cause for root canal therapy failure. Understanding of the bacterial content could improve treatment protocols. Fifty samples from 25 symptomatic and 25 asymptomatic previously root-filled teeth were collected from Sudanese patients with periradicular lesions. Amplified 16S rRNA gene (V1-V2) variable regions were subjected to pyrosequencing (FLX 454) to determine the bacterial profile. Obtained quality-controlled sequences from forty samples were classified into 741 operational taxonomic units (OTUs) at 3% dissimilarity, 525 at 5% dissimilarity and 297 at 10% dissimilarity, approximately corresponding to species-, genus- and class levels. The most abundant phyla were: Firmicutes (29.9%), Proteobacteria (26.1%), Actinobacteria (22.72%), Bacteroidetes (13.31%) and Fusobacteria (4.55%). Symptomatic patients had more Firmicutes and Fusobacteria than asymptomatic patients, while asymptomatic patients showed more Proteobacteria and Actinobacteria. Interaction of disease status and age was observed by two-way ANOSIM. Canonical correspondence analysis for age, tooth restoration and disease status showed a correlation of disease status with the composition and prevalence of different members of the microbial community. The pyrosequencing analysis revealed a distinctly higher diversity of the microbiota compared to earlier reports. The comparison of symptomatic and asymptomatic patients showed a clear association of the composition of the bacterial community with the presence and absence of symptoms in conjunction with the patients' age.

Background Although the importance and widespread occurrence of iron limitation in the contemporary ocean is well documented, we still know relatively little about genetic adaptation of phytoplankton to these environments. Compared to its coastal relative Thalassiosira pseudonana , the oceanic diatom Thalassiosira oceanica is highly tolerant to iron limitation. The adaptation to low-iron conditions in T. oceanica has been attributed to a decrease in the photosynthetic components that are rich in iron. Genomic information on T. oceanica may shed light on the genetic basis of the physiological differences between the two species. Results The complete 141790 bp sequence of the T. oceanica chloroplast genome [GenBank: GU323224], assembled from massively parallel pyrosequencing (454) shotgun reads, revealed that the petF gene encoding for ferredoxin, which is localized in the chloroplast genome in T. pseudonana and other diatoms, has been transferred to the nucleus in T. oceanica . The iron-sulfur protein ferredoxin, a key element of the chloroplast electron transport chain, can be replaced by the iron-free flavodoxin under iron-limited growth conditions thereby contributing to a reduction in the cellular iron requirements. From a comparison to the genomic context of the T. pseudonana petF gene, the T. oceanica ortholog can be traced back to its chloroplast origin. The coding potential of the T. oceanica chloroplast genome is comparable to that of T. pseudonana and Phaeodactylum tricornutum , though a novel expressed ORF appears in the genomic region that has been subjected to rearrangements linked to the petF gene transfer event. Conclusions The transfer of the petF from the cp to the nuclear genome in T. oceanica represents a major difference between the two closely related species. The ability of T. oceanica to tolerate iron limitation suggests that the transfer of petF from the chloroplast to the nuclear genome might have contributed to the ecological success of this species.

Key message Only few genetic loci are sufficient to increase the variation of bolting time in Beta vulgaris dramatically, regarding vernalization requirement, seasonal bolting time and reproduction type. Beta species show a wide variation of bolting time regarding the year of first reproduction, seasonal bolting time and the number of reproduction cycles. To elucidate the genetics of bolting time control, we used three F 3 mapping populations that were produced by crossing a semelparous, annual sugar beet with iteroparous, vernalization-requiring wild beet genotypes. The semelparous plants died after reproduction, whereas iteroparous plants reproduced at least twice. All populations segregated for vernalization requirement, seasonal bolting time and the number of reproduction cycles. We found that vernalization requirement co-segregated with the bolting locus B on chromosome 2 and was inherited independently from semel- or iteroparous reproduction. Furthermore, we found that seasonal bolting time is a highly heritable trait ( h 2  > 0.84), which is primarily controlled by two major QTL located on chromosome 4 and 9. Late bolting alleles of both loci act in a partially recessive manner and were identified in both iteroparous pollinators. We observed an additive interaction of both loci for bolting delay. The QTL region on chromosome 4 encompasses the floral promoter gene BvFT2 , whereas the QTL on chromosome 9 co-localizes with the BR 1 locus, which controls post-winter bolting resistance. Our findings are applicable for marker-assisted sugar beet breeding regarding early bolting to accelerate generation cycles and late bolting to develop bolting-resistant spring and winter beets. Unexpectedly, one population segregated also for dwarf growth that was found to be controlled by a single locus on chromosome 9.

Background Many hypothesis-driven genetic studies require the ability to comprehensively and efficiently target specific regions of the genome to detect sequence variations. Often, sample availability is limited requiring the use of whole genome amplification (WGA). We evaluated a high-throughput microdroplet-based PCR approach in combination with next generation sequencing (NGS) to target 384 discrete exons from 373 genes involved in cancer. In our evaluation, we compared the performance of six non-amplified gDNA samples from two HapMap family trios. Three of these samples were also preamplified by WGA and evaluated. We tested sample pooling or multiplexing strategies at different stages of the tested targeted NGS (T-NGS) workflow. Results The results demonstrated comparable sequence performance between non-amplified and preamplified samples and between different indexing strategies [sequence specificity of 66.0% ± 3.4%, uniformity (coverage at 0.2× of the mean) of 85.6% ± 0.6%]. The average genotype concordance maintained across all the samples was 99.5% ± 0.4%, regardless of sample type or pooling strategy. We did not detect any errors in the Mendelian patterns of inheritance of genotypes between the parents and offspring within each trio. We also demonstrated the ability to detect minor allele frequencies within the pooled samples that conform to predicted models. Conclusion Our described PCR-based sample multiplex approach and the ability to use WGA material for NGS may enable researchers to perform deep resequencing studies and explore variants at very low frequencies and cost.