Search

Phages integrated into a bacterial genome–called prophages–continuously monitor the health of the host bacteria to determine when to escape the genome, protect their host from other phage infections, and may provide genes that promote bacterial growth. Prophages are essential to almost all microbiomes, including the human microbiome. However, most human microbiome studies focus on bacteria, ignoring free and integrated phages, so we know little about how these prophages affect the human microbiome. We compared the prophages identified in 11,513 bacterial genomes isolated from human body sites to characterise prophage DNA in the human microbiome. Here, we show that prophage DNA comprised an average of 1-5% of each bacterial genome. The prophage content per genome varies with the isolation site on the human body, the health of the human, and whether the disease was symptomatic. The presence of prophages promotes bacterial growth and sculpts the microbiome. However, the disparities caused by prophages vary throughout the body.

Phages dominate every ecosystem on the planet. While virulent phages sculpt the microbiome by killing their bacterial hosts, temperate phages provide unique growth advantages to their hosts through lysogenic conversion. Many prophages benefit their host, and prophages are responsible for genotypic and phenotypic differences that separate individual microbial strains. However, the microbes also endure a cost to maintain those phages: additional DNA to replicate and proteins to transcribe and translate. We have never quantified those benefits and costs. Here, we analysed over two and a half million prophages from over half a million bacterial genome assemblies. Analysis of the whole dataset and a representative subset of taxonomically diverse bacterial genomes demonstrated that the normalised prophage density was uniform across all bacterial genomes above 2 Mbp. We identified a constant carrying capacity of phage DNA per bacterial DNA. We estimated that each prophage provides cellular services equivalent to approximately 2.4 % of the cell’s energy or 0.9 ATP per bp per hour. We demonstrate analytical, taxonomic, geographic, and temporal disparities in identifying prophages in bacterial genomes that provide novel targets for identifying new phages. We anticipate that the benefits bacteria accrue from the presence of prophages balance the energetics involved in supporting prophages. Furthermore, our data will provide a new framework for identifying phages in environmental datasets, diverse bacterial phyla, and from different locations.

Bacteroidota are the most common bacteria in the human gut and are responsible for degrading complex polysaccharides that would otherwise remain undigested. The abundance of Bacteroides in the gut is shaped by phages such as crAssphages that infect and kill them. While close to 600 genomes have been identified computationally, only four have been successfully cultured. Here, we identify and characterize three novel crAssphage species isolated from wastewater and infecting the bacterial hostBacteroides cellulosilyticusWH2. We named the novel species,Kehishuvirus winsdale(Bc01),Kolpuevirus frurule(Bc03), andRudgehvirus redwords(Bc11) which span two different families and three genera. These phages may not have co-evolved with their respective bacterial hosts. The phages had a conserved gene arrangement with known crAssphages, but gene similarity within phages belonging to the same taxa was highly variable. Across the three species, only two structural genes encoding a hypothetical protein and a tail spike protein were similar. Evolutionary analysis revealed the tail spike protein is undergoing purifying selection and was predicted to bind to a TonB-dependent transporter on the host cell surface, suggesting a role for host specificity. This study expands the known crAssphage isolates and reveals insights into the crAssphage infection mechanism. The availability of pure cultures of multiple crAssphage infecting the same host provides an opportunity to perform controlled experiments on one of the most dominant members of the human enteric virome.

BackgroundAnalysis of viral diversity using modern sequencing technologies offers extraordinary opportunities for discovery. However, these analyses present a number of bioinformatic challenges due to viral genetic diversity and virome complexity. Due to the lack of conserved marker sequences, metagenomic detection of viral sequences requires a non-targeted, random (shotgun) approach. Annotation and enumeration of viral sequences relies on rigorous quality control and effective search strategies against appropriate reference databases. Virome analysis also benefits from the analysis of both individual metagenomic sequences as well as assembled contigs. Combined, virome analysis results in large amounts of data requiring sophisticated visualization and statistical tools.ResultsHere we introduce Hecatomb, a bioinformatics platform enabling both read and contig based analysis. Hecatomb integrates query information from both amino acid and nucleotide reference sequence databases. Hecatomb integrates data collected throughout the workflow enabling analyst driven virome analysis and discovery. Hecatomb is available on GitHub at https://github.com/shandley/hecatomb.ConclusionsHecatomb provides a single, modular software solution to the complex tasks required of many virome analysis. We demonstrate the value of the approach by applying Hecatomb to both a host-associated (enteric) and an environmental (marine) virome data set. Hecatomb provided data to determine true- or false-positive viral sequences in both data sets and revealed complex virome structure at distinct marine reef sites.

Brettanomyces bruxellensis is considered one of the most problematic microbes associated with wine production. Sulfur dioxide is commonly used to inhibit the growth of B. bruxellensis and limit the potential wine spoilage. Brettanomyces bruxellensis wine isolates can grow at higher concentrations of this preservative than isolates from other sources. Thus, it has been suggested that the use of sulfite may have selected for B. bruxellensis strains better adapted to survive in the winemaking environment. We utilized laboratory adaptive evolution to determine the potential for this to occur. Three B. bruxellensis strains, representative of known genetic variation within the species, were subjected to increasing sublethal sulfur dioxide concentrations. Individual clones isolated from evolved populations displayed enhanced sulfite tolerance, ranging from 1.6 to 2.5 times higher than the corresponding parental strains. Whole-genome sequencing of sulfite-tolerant clones derived from two of the parental strains revealed structural variations affecting 270 genes. The region containing the sulfite efflux pump encoding gene, SSU1, showed clear copy number variants in all sequenced clones. Regardless of parental strain genetic background, SSU1 copy number changes were reproducibly associated with one SSU1 haplotype. This work clearly demonstrates adaptive evolution of B. bruxellensis when exposed to sublethal sulfites and suggests that, similar to Saccharomyces cerevisiae wine yeast, the mechanism responsible involves the gene SSU1.

Despite the increasing epidemiological evidence that the Onchocerca volvulus parasite is strongly associated with epilepsy in children, hence the name onchocerciasis-associated epilepsy (OAE), the pathophysiological mechanism of OAE remains to be elucidated. In June 2014, children with unprovoked convulsive epilepsy and healthy controls were enrolled in a case control study in Titule, Bas-Uélé Province in the Democratic Republic of the Congo (DRC) to identify risk factors for epilepsy. Using a subset of samples collected from individuals enrolled in this study (16 persons with OAE and 9 controls) plasma, buffy coat, and cerebrospinal fluid (CSF) were subjected to random-primed next-generation sequencing. The resulting sequences were analyzed using sensitive computational methods to identify viral DNA and RNA sequences. Anneloviridae, Flaviviridae, Hepadnaviridae (Hepatitis B virus), Herpesviridae, Papillomaviridae, Polyomaviridae (Human polyomavirus), and Virgaviridae were identified in cases and in controls. Not unexpectedly, a variety of bacteriophages were also detected in all cases and controls. However, none of the identified viral sequences were found enriched in OAE cases, which was our criteria for agents that might play a role in the etiology or pathogenesis of OAE.

Aureobasidium pullulans is the most abundant and ubiquitous species within the genus and is also considered a core component of the grape juice microflora. So far, a small number of other Aureobasidium species have been reported, that in contrast to A. pullulans, appear far more constrained to specific habitats. It is unknown whether grape juice is a reservoir of novel Aureobasidium species, overlooked in the course of conventional morphological and meta-barcoding analyses. In this study, eight isolates from grape juice taxonomically classified as Aureobasidium through ITS sequencing were subjected to whole-genome phylogenetic, synteny and nucleotide identity analyses, which revealed three isolates to likely represent newly discovered Aureobasidium species. Analyses of ITS and metagenomic sequencing datasets show that these species can be present in grape juice samples from different locations and vintages. Functional annotation revealed the Aureobasidium isolates possess the genetic potential to support growth on the surface of plants and grapes. However, the loss of several genes associated with tolerance to diverse environmental stresses suggest a more constrained ecological range than A. pullulans.

Background Most bacterial genomes contain integrated bacteriophages—prophages—in various states of decay. Many are active and able to excise from the genome and replicate, while others are cryptic prophages, remnants of their former selves. Over the last two decades, many computational tools have been developed to identify the prophage components of bacterial genomes, and it is a particularly active area for the application of machine learning approaches. However, progress is hindered and comparisons thwarted because there are no manually curated bacterial genomes that can be used to test new prophage prediction algorithms. Methods We present a library of gold-standard bacterial genome annotations that include manually curated prophage annotations, and a computational framework to compare the predictions from different algorithms. We use this suite to compare all extant stand-alone prophage prediction algorithms to identify their strengths and weaknesses. We provide a FAIR dataset for prophage identification, and demonstrate the accuracy, precision, recall, and f1 score from the analysis of seven different algorithms for the prediction of prophages. Results We identified different strengths and weaknesses between the prophage prediction tools. Several tools exhibit exceptional f1 scores, while others have better recall at the expense of more false positives. The tools vary greatly in runtime performance with few exhibiting all desirable qualities for large-scale analyses. Conclusions Our library of gold-standard prophage annotations and benchmarking framework provide a valuable resource for exploring strengths and weaknesses of current and future prophage annotation tools. We discuss caveats and concerns in this analysis, how those concerns may be mitigated, and avenues for future improvements. This framework will help developers identify opportunities for improvement and test updates. It will also help users in determining the tools that are best suited for their analysis.

The addition of SO(2) is practiced in the wine industry to mitigate the risk of microbial spoilage and to extend wine shelf-life. Generally, this strategy does not interfere with primary alcoholic fermentation, as wine strains of Saccharomyces cerevisiae exhibit significant SO(2) tolerance, largely driven by the efflux pump Ssu1p. One of the key yeast species responsible for wine spoilage is Brettanomyces bruxellensis, which also exhibits strain-dependent SO(2) tolerance, although this occurs via unknown mechanisms. To evaluate the factors responsible for the differential sulfite tolerance observed in B. bruxellensis strains, we employed a multifaceted approach to examine both expression and allelic differences in the BbSSU1 gene. Transcriptomic analysis following exposure to SO(2) highlighted different inducible responses in two B. bruxellensis strains. It also revealed disproportionate transcription of one putative BbSSU1 haplotype in both genetic backgrounds. Here, we confirm the functionality of BbSSU1 by complementation of a null mutant in a S. cerevisiae wine strain. The expression of four distinct BbSSU1 haplotypes in the S. cerevisiae ΔSSU1 mutant revealed up to a 3-fold difference in conferred SO(2) tolerance. Substitution of key amino acids distinguishing the encoded proteins was performed to evaluate their relative contribution to SO(2) tolerance. Protein modeling of two haplotypes which differed in two amino acid residues suggested that these substitutions affect the binding of Ssu1p ligands near the channel opening. Taken together, preferential transcription of a BbSSU1 allele that encodes a more efficient Ssu1p transporter may represent one mechanism that contributes to differences in sulfite tolerances between B. bruxellensis strains. IMPORTANCE Brettanomyces bruxellensis is one of the most important wine spoilage microorganisms, with the use of sulfite being the major method to control spoilage. However, this species displays a wide intraspecies distribution in sulfite tolerance, with some strains capable of tolerating high concentrations of SO(2), with relatively high concentrations of this antimicrobial needed for their control. Although SO(2) tolerance has been studied in several organisms and particularly in S. cerevisiae, little is known about the mechanisms that confer SO(2) tolerance in B. bruxellensis. Here, we confirmed the functionality of the sulfite efflux pump encoded by BbSSU1 and determined the efficiencies of four different BbSSU1 haplotypes. Gene expression analysis showed greater expression of the haplotype conferring greater SO(2) tolerance. Our results suggest that a combination of BbSSU1 haplotype efficiency, copy number, and haplotype expression levels likely contributes to the diverse SO(2) tolerances observed for different B. bruxellensis strains.

Chardonnay is the basis of some of the world’s most iconic wines and its success is underpinned by a historic program of clonal selection. There are numerous clones of Chardonnay available that exhibit differences in key viticultural and oenological traits that have arisen from the accumulation of somatic mutations during centuries of asexual propagation. However, the genetic variation that underlies these differences remains largely unknown. To address this knowledge gap, a high-quality, diploid-phased Chardonnay genome assembly was produced from single-molecule real time sequencing, and combined with re-sequencing data from 15 different Chardonnay clones. There were 1620 markers identified that distinguish the 15 clones. These markers were reliably used for clonal identification of independently sourced genomic material, as well as in identifying a potential genetic basis for some clonal phenotypic differences. The predicted parentage of the Chardonnay haplomes was elucidated by mapping sequence data from the predicted parents of Chardonnay (Gouais blanc and Pinot noir) against the Chardonnay reference genome. This enabled the detection of instances of heterosis, with differentially-expanded gene families being inherited from the parents of Chardonnay. Most surprisingly however, the patterns of nucleotide variation present in the Chardonnay genome indicate that Pinot noir and Gouais blanc share an extremely high degree of kinship that has resulted in the Chardonnay genome displaying characteristics that are indicative of inbreeding., Author summary Phenotypic variation within a grapevine cultivar arises from an accumulation of mutations from serial vegetative propagation. Old cultivars such as Chardonnay have been propagated for centuries resulting in hundreds of available ‘clones’ containing unique genetic mutations and a range of various phenotypic peculiarities. The genetic mutations can be leveraged as genetic markers and are useful in identifying specific clones for authenticity testing, or as breeding markers for new clonal selections where particular mutations are known to confer a phenotypic trait. We produced a high-quality genome assembly for Chardonnay, and using re-sequencing data for 15 popular clones, were able to identify a large selection of markers that are unique to at least one clone. We identified mutations that may confer phenotypic effects, and were able to identify clones from material independently sourced from nurseries and vineyards. The marker detection framework we describe for authenticity testing would be applicable to other grapevine cultivars or even other agriculturally important woody-plant crops that are vegetatively propagated such as fruit orchards. Finally, we show that the Chardonnay genome contains extensive evidence for parental inbreeding, such that its parents, Gouais blanc and Pinot noir, may even represent first-degree relatives.

Availability of a reliable and consistent source for high temperature ceramic capacitors has been an ongoing issue for design engineers looking at developing product for applications up to and including +300°C. The general practice has been to derate standard X7R ceramic capacitors for high temperature applications and settle for a device characterized by a significant reduction in those critical performance features related to capacitance value, operating voltage, insulation resistance and breakdown voltage, not to mention a substantial roll off in temperature coefficient above +150°C. In addition, the need to provide coated and / or leaded options also presents additional concerns related to operational integrity. This paper presents packaging options, life test reliability data and compares performance characteristics for a unique high K, high temperature ceramic capacitor, to the more traditional options. This approach utilizes leading edge, Class II dielectric and packaging materials that have been specifically developed for use at +300°C and then benefit from enhanced reliability when operated at lower temperatures. As shown in this paper, capacitors manufactured with this dielectric material exhibit much higher capacitance per unit volume and significant improvements in insulation resistance, without having to sacrifice mechanical strength, voltage rating or long term reliability.