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Since Antonie van Leeuwenhoek first observed microbes, we have come to learn that life on our planet includes not only the macroscopic lifeforms visible to the unaided eye that Charles Darwin studied but also an immense diversity of microbes (Fig 1). To reflect the importance of microbes and to create a home for microbiology research using genetics, I PLOS Genetics i will be launching a new section on Microbial Genetics. Genetic systems have been established in many microbes, and the advent of omics technologies allows studying microbes without established genetic systems. [Extracted from the article]

Social network analysis has been postulated as a tool to study potential pathogen transmission in wildlife but is resource‐intensive to quantify. Networks based on bacterial genotypes have been proposed as a cost‐effective method for estimating social or transmission network based on the assumption that individuals in close contact will share commensal bacteria. However, the use of network analysis to study wild populations requires critical evaluation of the assumptions and parameters these models are founded on.We test (a) whether networks of commensal bacterial sharing are related to hosts' social associations and hence could act as a proxy for estimating transmission networks, (b) how the parameters chosen to define host associations and delineate bacterial genotypes impact inference and (c) whether these relationships change across time. We use stochastic simulations to evaluate how uncertainty in parameter choice affects network structure.We focused on a well‐studied population of eastern grey kangaroos (Macropus giganteus), from Sundown National Park, Australia. Using natural markings, each individual was identified and its associations with other kangaroos recorded through direct field observations over 2 years to construct social networks. Faecal samples were collected, Escherichia coli was cultured and genotyped using BOX‐PCR, and bacterial networks were constructed. Two individuals were connected in the bacterial network if they shared at least one E. coli genotype. We determined the capacity of bacterial networks to predict the observed social network structure in each year.We found little support for a relationship between social association and dyadic commensal bacterial similarity. Thresholds to determine host associations and similarity cut‐off values used to define E. coli genotypes had important ramifications for inferring links between individuals. In fact, we found that inferences can show opposite patterns based on the chosen thresholds. Moreover, no similarity in overall bacterial network structure was detected between years.Although empirical disease transmission data are often unavailable in wildlife populations, both bacterial networks and social networks have limitations in representing the mode of transmission of a pathogen. Our results suggest that caution is needed when designing such studies and interpreting results. [ABSTRACT FROM AUTHOR]

The incidence of colorectal cancer (CRC) is gradually rising in sub-Saharan Africa. This may be due to dietary changes associated with urbanization, which may induce tumor-promoting gut microbiota composition and function. We compared fecal microbiota composition and activity in 10 rural and 10 urban Zimbabweans for evidence of a differential CRC risk. Dietary intake was assessed by a food frequency questionnaire. Fecal microbiota composition, metabolomic profile, functional microbial genes were analyzed, and bile acids and short chain fatty acids quantified. Animal protein intake was higher among urban volunteers, but carbohydrate and fiber intake were similar. Bacteria related to Blautia obeum, Streptococcus bovis, and Subdoligranulum variabile were higher in urban residents, whereas bacteria related to Oscillospira guillermondii and Sporobacter termitidis were higher in rural volunteers. Fecal levels of primary bile acids, cholic acid, and chenodeoxycholic acid (P < 0.05), and secondary bile acids, deoxycholic acid (P < 0.05) and ursodeoxycholic acid (P < 0.001) were higher in urban residents. Fecal levels of acetate and propionate, but not butyrate, were higher in urban residents. The gut microbiota composition and activity among rural and urban Zimbabweans retain significant homogeneity (possibly due to retention of dietary fiber), but urban residents have subtle changes, which may indicate a higher CRC risk. [ABSTRACT FROM AUTHOR]

The humoral immune responses of the nipa palm hispid beetle Octodonta nipae involves the inducible expression of the genes coding for antimicrobial peptides (AMPs) which are mediated by immune deficiency signaling pathways. In insects, the nuclear factor-κB (NF−κB) transcription factor, Relish, has been shown to regulate AMP gene expressions upon microbial infections. Here, we dissect the expression patterns of some AMPs in O. nipae during infections by entomopathogenic nematodes (EPNs) and their symbionts, before and after Relish knock down. Our results indicate that, prior to gene silencing, the AMPs attacin C1, attacin C2, and defensin 2B were especially expressed to great extents in the insects challenged with the nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora as well as with their respective symbionts Xenorhabdus nematophila and Photorhabdus luminescens. The study also established the partial sequence of OnRelish/NF-κB p110 subunit in O. nipae , with an open reading frame coding for a protein with 102 amino acid residues. A typical Death domain-containing protein was detected (as seen in Drosophila) at the C-terminus of the protein. Phylogenetic analysis revealed that in O. nipae , Relish is clustered with registered Relish/NF-κB p110 proteins from other species of insect especially Leptinotarsa decemlineata from the same order Coleoptera. Injection of OnRelish dsRNA remarkably brought down the expression of OnRelish and also reduced the magnitude of transcription of attacin C1 and defensin 2B upon S. carpocapsae and H. bacteriophora and their symbionts infections. Altogether, our data unveil the expression pattern of OnRelish as well as that of some AMP genes it influences during immune responses of O. nipae against EPNs and their symbionts. [ABSTRACT FROM AUTHOR]

Background: Prophages play a significant role in prokaryotic evolution, often altering the function of the cell that they infect via transfer of new genes e.g., virulence or antibiotic resistance factors, inactivation of existing genes or by modifying gene expression. Recently, phage therapy has gathered renewed interest as a promising alternative to control bacterial infections. Cataloging the repertoire of prophages in large collections of species’ genomes is an important initial step in understanding their evolution and potential therapeutic utility. However, current widely-used tools for identifying prophages within bacterial genome sequences are mainly web-based, can have long response times, and do not scale to keep pace with the many thousands of genomes currently being sequenced routinely. Methodology: In this work, we present ProphET, an easy to install prophage predictor to be used in Linux operation system, without the constraints associated with a web-based tool. ProphET predictions rely on similarity searches against a database of prophage genes, taking as input a bacterial genome sequence in FASTA format and its corresponding gene annotation in GFF. ProphET identifies prophages in three steps: similarity search, calculation of the density of prophage genes, and edge refinement. ProphET performance was evaluated and compared with other phage predictors based on a set of 54 bacterial genomes containing 267 manually annotated prophages. Findings and conclusions: ProphET identifies prophages in bacterial genomes with high precision and offers a fast, highly scalable alternative to widely-used web-based applications for prophage detection. [ABSTRACT FROM AUTHOR]

Introduction: Mucosal immune activation, in the context of sexual transmission of HIV-1 infection, is crucial, as the increased presence of activated T cells enhance susceptibility to infection. In this regard, it has been proposed that immunomodulatory compounds capable of modulating immune activation, such as Vitamin D (VitD) may reduce HIV-1 transmission and might be used as a safe and cost-effective strategy for prevention. Considering this, we examined the in vitro effect of the treatment of peripheral blood mononuclear cells (PBMCs) with the active form of VitD, calcitriol, on cellular activation, function and susceptibility of CD4+ T cells to HIV-1 infection. Methods: We treated PBMCs from healthy HIV unexposed individuals (Co-HC) and frequently exposed, HIV-1 seronegative individuals (HESNs) from Colombia and from healthy non-exposed individuals from Canada (Ca-HC) with calcitriol and performed in vitro HIV-1 infection assays using X4- and R5-tropic HIV-1 strains respectively. In addition, we evaluated the activation and function of T cells and the expression of viral co-receptors, and select antiviral genes following calcitriol treatment. Results: Calcitriol reduced the frequency of infected CD4+ T cells and the number of viral particles per cell, for both, X4- and R5-tropic viruses tested in the Co-HC and the Ca-HC, respectively, but not in HESNs. Furthermore, in the Co-HC, calcitriol reduced the frequency of polyclonally activated T cells expressing the activation markers HLA-DR and CD38, and those HLA-DR+CD38-, whereas increased the subpopulation HLA-DR-CD38+. Calcitriol treatment also decreased production of granzyme, IL-2 and MIP-1β by T cells and increased the transcriptional expression of the inhibitor of NF-kB and the antiviral genes cathelicidin (CAMP) and APOBEC3G in PBMCs from Co-HC. Conclusion: Our in vitro findings suggest that VitD treatment could reduce HIV-1 transmission through a specific modulation of the activation levels and function of T cells, and the production of antiviral factors. In conclusion, VitD remains as an interesting potential strategy to prevent HIV-1 transmission that should be further explored. [ABSTRACT FROM AUTHOR]

Errors throughout gene expression are likely deleterious, hence genomes are under selection to ameliorate their consequences. Additional stop codons (ASCs) are in-frame nonsense ‘codons’ downstream of the primary stop which may be read by translational machinery should the primary stop have been accidentally read through. Prior evidence in several eukaryotes suggests that ASCs are selected to prevent potentially-deleterious consequences of read-through. We extend this evidence showing that enrichment of ASCs is common but not universal for single cell eukaryotes. By contrast, there is limited evidence as to whether the same is true in other taxa. Here, we provide the first systematic test of the hypothesis that ASCs act as a fail-safe mechanism in eubacteria, a group with high read-through rates. Contra to the predictions of the hypothesis we find: there is paucity, not enrichment, of ASCs downstream; substitutions that degrade stops are more frequent in-frame than out-of-frame in 3’ sequence; highly expressed genes are no more likely to have ASCs than lowly expressed genes; usage of the leakiest primary stop (TGA) in highly expressed genes does not predict ASC enrichment even compared to usage of non-leaky stops (TAA) in lowly expressed genes, beyond downstream codon +1. Any effect at the codon immediately proximal to the primary stop can be accounted for by a preference for a T/U residue immediately following the stop, although if anything, TT- and TC- starting codons are preferred. We conclude that there is no compelling evidence for ASC selection in eubacteria. This presents an unusual case in which the same error could be solved by the same mechanism in eukaryotes and prokaryotes but is not. We discuss two possible explanations: that, owing to the absence of nonsense mediated decay, bacteria may solve read-through via gene truncation and in eukaryotes certain prion states cause raised read-through rates. [ABSTRACT FROM AUTHOR]

Unknown sequences, or gaps, are present in many published genomes across public databases. Gap filling is an important finishing step in de novo genome assembly, especially in large genomes. The gap filling problem is nontrivial and while there are many computational tools partially solving the problem, several have shortcomings as to the reliability and correctness of the output, i.e. the gap filled draft genome. SSPACE-LongRead is a scaffolding tool that utilizes long reads from multiple third-generation sequencing platforms in finding links between contigs and combining them. The long reads potentially contain sequence information to fill the gaps created in the scaffolding, but SSPACE-LongRead currently lacks this functionality. We present an automated pipeline called gapFinisher to process SSPACE-LongRead output to fill gaps after the scaffolding. gapFinisher is based on the controlled use of a previously published gap filling tool FGAP and works on all standard Linux/UNIX command lines. We compare the performance of gapFinisher against two other published gap filling tools PBJelly and GMcloser. We conclude that gapFinisher can fill gaps in draft genomes quickly and reliably. In addition, the serial design of gapFinisher makes it scale well from prokaryote genomes to larger genomes with no increase in the computational footprint. [ABSTRACT FROM AUTHOR]

Host-microbiota interactions describe a co-evolution and mutualistic symbiosis. Gut microbial communities are important for diverse host functions. However, in birds, the relationship between the composition of the intestinal microbiota and the genetic variation of the host is not clearly understood. To dissect these interactions, a Chinese yellow broiler line (genetically selected for a high growth rate) and Huiyang Beard chickens (low growth rate) were crossed, generating an F2 population. The population structures of the gut microbes in the phenotypically high and low 91-d body weight individuals of both sexes in the F2 population were studied. Interestingly, a non-metric multidimensional scaling analysis revealed that the microbiota of the high-weight and low-weight females was clearly separated into 2 clusters. A β-diversity analysis showed that the locus rs16775833 within the doublesex and mab-3-related transcription factor (DMRT) gene cluster accounted for approximately 21% of the variation in the population structure of the gut microbiota. Furthermore, the 2 genetic loci rs15142709 and rs15142674 were significantly associated with specific species of Methanobacterium. These loci are located in the pleiomorphic adenoma gene 1 (PLAG1) and lck/yes-related novel tyrosine kinase (LYN) genes, which are involved in cell differentiation and growth. This finding suggests evidence for the influence of the host genetics on the composition of the gut microbiota in birds and the importance and utility of the host-microbe status to better understand its effect on the potential growth of birds. [ABSTRACT FROM AUTHOR]

Anorexia nervosa (AN) has one of the highest mortality rates of any psychiatric disorder. Treatments are often ineffective and relapse is common. Most research attempting to understand the underlying causes and maintenance factors of AN has focused on environmental contributions, yet there is much to be explored in terms of biological risk and maintenance factors. In this paper, we focus primarily on AN research related to genetics and the complex microbial community in the gut (intestinal microbiota), and how these impact our conceptualization of this disorder. Emerging research identifying significant negative genetic correlations between AN and obesity suggests that the conditions may represent 'metabolic bookends'. The identification of underlying biological mechanisms may provide both insight into extreme weight dysregulation on both ends of the spectrum and new possible points of entry for AN treatment. Additionally, the reported microbial imbalance (dysbiosis) in the gut microbiota in AN patients, potentially due to a nutrient‐ and energy‐deprived gut environment, implies alterations in functional and metabolic capacity of the gut microbiome. The extent to which AN and obesity can also be considered to be 'microbiome bookends' requires further investigation. Finally, we discuss ongoing and future AN projects exploring the interplay between host genomics, the environment, and cumulative microbial genomes (microbiome) as well as interventions at the microbial and gut level. [ABSTRACT FROM AUTHOR]

Using metagenomics analysis, we are the first to identify the presence of a small, circular, single-stranded Gemykibivirus (GkV) genome from the respiratory tract of an elderly woman with severe acute respiratory distress syndrome. Our results suggest that further studies on whether GkVs infect humans and cause respiratory disease are needed. [ABSTRACT FROM AUTHOR]

Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe. H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2+--a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of ams2+ antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9--an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)--indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 proteincoding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycledependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression. [ABSTRACT FROM AUTHOR]

The fission yeast Schizosaccharomyces pombe is a powerful genetic model system for uncovering fundamental principles of heterochromatin assembly and epigenetic inheritance of chromatin states. Heterochromatin defined by histone H3 lysine 9 methylation and HP1 proteins coats large chromosomal domains at centromeres, telomeres, and the mating-type (mat) locus. Although genetic and biochemical studies have provided valuable insights into heterochromatin assembly, many key mechanistic details remain unclear. Here, we use a sensitized reporter system at the mat locus to screen for factors affecting heterochromatic silencing. In addition to known components of heterochromatin assembly pathways, our screen identified eight new factors including the cohesin-associated protein Pds5. We find that Pds5 enriched throughout heterochromatin domains is required for proper maintenance of heterochromatin. This function of Pds5 requires its associated Eso1 acetyltransferase, which is implicated in the acetylation of cohesin. Indeed, introducing an acetylation-mimicking mutation in a cohesin subunit suppresses defects in heterochromatin assembly in pds5D and eso1D cells. Our results show that in cells lacking Pds5, cohesin interferes with heterochromatin assembly. Supporting this, eliminating cohesin from the mat locus in the pds5D mutant restores both heterochromatin assembly and gene silencing. These analyses highlight an unexpected requirement for Pds5 in ensuring proper coordination between cohesin and heterochromatin factors to effectively maintain gene silencing. [ABSTRACT FROM AUTHOR]

Maintenance of genome integrity is a crucial cellular focus that involves a wide variety of proteins functioning in multiple processes. Defects in many different pathways can result in genome instability, a hallmark of cancer. Utilizing a diploid Saccharomyces cerevisiae model, we previously reported a collection of gene mutations that affect genome stability in a haploinsufficient state. In this work we explore the effect of gene dosage on genome instability for one of these genes and its paralog; SAM1 and SAM2. These genes encode S-Adenosylmethionine (AdoMet) synthetases, responsible for the creation of AdoMet from methionine and ATP. AdoMet is the universal methyl donor for methylation reactions and is essential for cell viability. It is the second most used cellular enzyme substrate and is exceptionally well-conserved through evolution. Mammalian cells express three genes, MAT1A, MAT2A, and MAT2B, with distinct expression profiles and functions. Alterations to these AdoMet synthetase genes, and AdoMet levels, are found in many cancers, making them a popular target for therapeutic intervention. However, significant variance in these alterations are found in different tumor types, with the cellular consequences of the variation still unknown. By studying this pathway in the yeast system, we demonstrate that losses of SAM1 and SAM2 have different effects on genome stability through distinctive effects on gene expression and AdoMet levels, and ultimately separate effects on the methyl cycle. Thus, this study provides insight into the mechanisms by which differential expression of the SAM genes have cellular consequences that affect genome instability. [ABSTRACT FROM AUTHOR]

The microbial communities that inhabit the distal gut of humans and other mammals exhibit large inter-individual variation. While host genetics is a known factor that influences gut microbiota composition, the mechanisms underlying this variation remain largely unknown. Bile acids (BAs) are hormones that are produced by the host and chemically modified by gut bacteria. BAs serve as environmental cues and nutrients to microbes, but they can also have antibacterial effects. We hypothesized that host genetic variation in BA metabolism and homeostasis influence gut microbiota composition. To address this, we used the Diversity Outbred (DO) stock, a population of genetically distinct mice derived from eight founder strains. We characterized the fecal microbiota composition and plasma and cecal BA profiles from 400 DO mice maintained on a high-fat high-sucrose diet for ~22 weeks. Using quantitative trait locus (QTL) analysis, we identified several genomic regions associated with variations in both bacterial and BA profiles. Notably, we found overlapping QTL for Turicibacter sp. and plasma cholic acid, which mapped to a locus containing the gene for the ileal bile acid transporter, Slc10a2. Mediation analysis and subsequent follow-up validation experiments suggest that differences in Slc10a2 gene expression associated with the different strains influences levels of both traits and revealed novel interactions between Turicibacter and BAs. This work illustrates how systems genetics can be utilized to generate testable hypotheses and provide insight into host-microbe interactions. [ABSTRACT FROM AUTHOR]

Almost one third of herpesvirus proteins are expressed with late kinetics. Many of these late proteins serve crucial structural functions such as formation of virus particles, attachment to host cells and internalization. Recently, we and others identified a group of Epstein-Barr virus early proteins that form a pre-initiation complex (vPIC) dedicated to transcription of late genes. Currently, there is a fundamental gap in understanding the role of post-translational modifications in regulating assembly and function of the complex. Here, we used mass spectrometry to map potential phosphorylation sites in BGLF3, a core component of the vPIC module that connects the BcRF1 viral TATA box binding protein to other components of the complex. We identified threonine 42 (T42) in BGLF3 as a phosphoacceptor residue. T42 is conserved in BGLF3 orthologs encoded by other gamma herpesviruses. Abolishing phosphorylation at T42 markedly reduced expression of vPIC-dependent late genes and disrupted production of new virus particles, but had no effect on early gene expression, viral DNA replication, or expression of vPIC-independent late genes. We complemented failure of BGLF3(T42A) to activate late gene expression by ectopic expression of other components of vPIC. Only BFRF2 and BVLF1 were sufficient to suppress the defect in late gene expression associated with BGLF3(T42A). These results were corroborated by the ability of wild type BGLF3 but not BGLF3(T42A) to form a trimeric complex with BFRF2 and BVLF1. Our findings suggest that phosphorylation of BGLF3 at threonine 42 serves as a new checkpoint for subsequent formation of BFRF2:BGLF3:BVLF1; a trimeric subcomplex essential for transcription of late genes. Our findings provide evidence that post-translational modifications regulate the function of the vPIC nanomachine that initiates synthesis of late transcripts in herpesviruses. [ABSTRACT FROM AUTHOR]

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis. [ABSTRACT FROM AUTHOR]

Model-based phylodynamic approaches recently employed generalized linear models (GLMs) to uncover potential predictors of viral spread. Very recently some of these models have allowed both the predictors and their coefficients to be time-dependent. However, these studies mainly focused on predictors that are assumed to be constant through time. Here we inferred the phylodynamics of avian influenza A virus H9N2 isolated in 12 Asian countries and regions under both discrete trait analysis (DTA) and structured coalescent (MASCOT) approaches. Using MASCOT we applied a new time-dependent GLM to uncover the underlying factors behind H9N2 spread. We curated a rich set of time-series predictors including annual international live poultry trade and national poultry production figures. This time-dependent phylodynamic prediction model was compared to commonly employed time-independent alternatives. Additionally the time-dependent MASCOT model allowed for the estimation of viral effective sub-population sizes and their changes through time, and these effective population dynamics within each country were predicted by a GLM. International annual poultry trade is a strongly supported predictor of virus migration rates. There was also strong support for geographic proximity as a predictor of migration rate in all GLMs investigated. In time-dependent MASCOT models, national poultry production was also identified as a predictor of virus genetic diversity through time and this signal was obvious in mainland China. Our application of a recently introduced time-dependent GLM predictors integrated rich time-series data in Bayesian phylodynamic prediction. We demonstrated the contribution of poultry trade and geographic proximity (potentially unheralded wild bird movements) to avian influenza spread in Asia. To gain a better understanding of the drivers of H9N2 spread, we suggest increased surveillance of the H9N2 virus in countries that are currently under-sampled as well as in wild bird populations in the most affected countries. [ABSTRACT FROM AUTHOR]

Generalized transduction, discovered in I Salmonella i phage P22, was the first mechanism of phage-mediated gene transfer to be identified [[8]]. However, this theory turned out to be inaccurate because phages capable of generalized transduction are not known to cause the breakdown of the bacterial chromosome into phage-sized or smaller DNA fragments [[9], [10]]. Finally, to offset the predictably catastrophic effects (splitting the viral genome in two) of in situ DNA packaging, in situ theta replication creates multiple integrated genomes so that both in situ DNA packaging and phage maturation can proceed in parallel. Phage terminase initiates DNA packaging from phage pac sites (black arrows) or phage pseudo-pac sites (gray arrows). [Extracted from the article]

Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in Malassezia furfur. A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10. This system is based on cotransformation of M. furfur mediated by A. tumefaciens, to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur, through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion. [ABSTRACT FROM AUTHOR]

Whole genome sequencing can provide essential public health information. However, it is now known that widely used short-read methods have the potential to miss some randomly-distributed segments of genomes. This can prevent phages, plasmids, and virulence factors from being detected or properly identified. Here, we compared assemblies of three complete Shiga toxin-producing Escherichia coli (STEC) O26:H11/H- genomes from two different sequence types (ST21 and 29), each acquired using the Nextera XT MiSeq, MinION nanopore-based sequencing, and Pacific Biosciences (PacBio) sequencing. Each closed genome consisted of a single chromosome, approximately 5.7 Mb for CFSAN027343, 5.6 Mb for CFSAN027346, and 5.4 MB for CFSAN027350. However, short-read whole genome sequencing (WGS) using Nextera XT MiSeq failed to identify some virulence genes in plasmids and on the chromosome, both of which were detected using the long-read platforms. Results from long-read MinION and PacBio allowed us to identify differences in plasmid content: a single 88 kb plasmid in CFSAN027343; a 157kb plasmid in CFSAN027350; and two plasmids in CFSAN027346 (one 95 Kb, one 72 Kb). These data enabled rapid characterization of the virulome, detection of antimicrobial genes, and composition/location of Stx phages. Taken together, positive correlations between the two long-read methods for determining plasmids, virulome, antimicrobial resistance genes, and phage composition support MinION sequencing as one accurate and economical option for closing STEC genomes and identifying specific virulence markers. [ABSTRACT FROM AUTHOR]

The 2017–2018 influenza epidemic season in Russia was characterized by a relatively low morbidity and mortality. We evaluated herd immunity prior to the 2017–2018 influenza season in hemagglutination inhibition assay, and performed characterization of influenza viruses isolated from severe or fatal influenza cases and from influenza cases in people vaccinated in the fall of 2017. During the 2017–2018 epidemic season, 87 influenza A and B viruses were isolated and viruses of the 75 influenza cases, including selected viral isolates and viruses analyzed directly from the original clinical material, were genetically characterized. The analyzed A(H1N1)pdm09 viruses belonged to clade 6B.1, B/Yamagata-like viruses belonged to clade 3, and B/Victoria-like viruses belonged to clade 1A and they were antigenically similar to the corresponding vaccine strains. A(H3N2) viruses belonged to clade 3C.2a and were difficult to characterize antigenically and the analysis indicated antigenic differences from the corresponding egg-grown vaccine strain. The next generation sequencing revealed the presence of D222/G/N polymorphism in the hemagglutinin gene in 32% of the analyzed A(H1N1)pdm09 lethal cases. This study demonstrated the importance of monitoring D222G/N polymorphism, including detection of minor viral variants with the mutations, in the hemagglutinin gene of A(H1N1)pdm09 for epidemiological surveillance. One strain of influenza virus A(H1N1)pdm09 was resistant to oseltamivir and had the H275Y amino acid substitution in the NA protein. All other isolates were susceptible to NA inhibitors. Prior to the 2017–2018 epidemic season, 67.4 million people were vaccinated, which accounted for 46.6% of the country's population. Just before the epidemic season 33–47% and 24–30% of blood sera samples collected within the territory of Russia showed the presence of protective antibody titers against vaccine strains of influenza A and influenza B/Victoria-like, respectively. Mass vaccination of the population had evidently reduced the severity of the flu epidemic during the 2017–2018 influenza epidemic season in Russia. [ABSTRACT FROM AUTHOR]

The spatial organization of DNA is mediated by the Par protein system in some bacteria. ParB binds specifically to the parS sequence on DNA and orchestrates its motion by interacting with ParA bound to the nucleoid. In the case of plasmids, a single ParB bound plasmid is observed to execute oscillations between cell poles while multiple plasmids eventually settle at equal distances from each other along the cell’s length. While the potential mechanism underlying the ParA-ParB interaction has been discussed, it remains unclear whether ParB-complex oscillations are stable limit cycles or merely decaying transients to a fixed point. How are dynamics affected by substrate length and the number of complexes? We present a deterministic model for ParA-ParB driven DNA segregation where the transition between stable arrangements and oscillatory behaviour depends only on five parameters: ParB-complex number, substrate length, ParA concentration, ParA hydrolysis rate and the ratio of the lengthscale over which the ParB complex stimulates ParA hydrolysis to the lengthscale over which ParA interacts with the ParB complex. When the system is buffered and the ParA rebinding rate is constant we find that ParB-complex dynamics is independent of substrate length and complex number above a minimum system size. Conversely, when ParA resources are limited, we find that changing substrate length and increasing complex number leads to counteracting mechanisms that can both generate or subdue oscillatory dynamics. We argue that cells may be poised near a critical level of ParA so that they can transition from oscillatory to fixed point dynamics as the cell cycle progresses so that they can both measure their size and faithfully partition their genetic material. Lastly, we show that by modifying the availability of ParA or depletion zone size, we can capture some of the observed differences in ParB-complex positioning between replicating chromosomes in B. subtilis cells and low-copy plasmids in E. coli cells. [ABSTRACT FROM AUTHOR]

Determining the fitness of specific microbial genotypes has extensive application in microbial genetics, evolution, and biotechnology. While estimates from growth curves are simple and allow high throughput, they are inaccurate and do not account for interactions between costs and benefits accruing over different parts of a growth cycle. For this reason, pairwise competition experiments are the current "gold standard" for accurate estimation of fitness. However, competition experiments require distinct markers, making them difficult to perform between isolates derived from a common ancestor or between isolates of nonmodel organisms. In addition, competition experiments require that competing strains be grown in the same environment, so they cannot be used to infer the fitness consequence of different environmental perturbations on the same genotype. Finally, competition experiments typically consider only the end-points of a period of competition so that they do not readily provide information on the growth differences that underlie competitive ability. Here, we describe a computational approach for predicting density-dependent microbial growth in a mixed culture utilizing data from monoculture and mixed-culture growth curves. We validate this approach using 2 different experiments with Escherichia coli and demonstrate its application for estimating relative fitness. Our approach provides an effective way to predict growth and infer relative fitness in mixed cultures. [ABSTRACT FROM AUTHOR]

Temperate phages are bacterial viruses that as part of their life cycle reside in the bacterial genome as prophages. They are found in many species including most clinical strains of the human pathogens, Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Previously, temperate phages were considered as only bacterial predators, but mounting evidence point to both antagonistic and mutualistic interactions with for example some temperate phages contributing to virulence by encoding virulence factors. Here we show that generalized transduction, one type of bacterial DNA transfer by phages, can create conditions where not only the recipient host but also the transducing phage benefit. With antibiotic resistance as a model trait we used individual-based models and experimental approaches to show that antibiotic susceptible cells become resistant to both antibiotics and phage by i) integrating the generalized transducing temperate phages and ii) acquiring transducing phage particles carrying antibiotic resistance genes obtained from resistant cells in the environment. This is not observed for non-generalized transducing temperate phages, which are unable to package bacterial DNA, nor for generalized transducing virulent phages that do not form lysogens. Once established, the lysogenic host and the prophage benefit from the existence of transducing particles that can shuffle bacterial genes between lysogens and for example disseminate resistance to antibiotics, a trait not encoded by the phage. This facilitates bacterial survival and leads to phage population growth. We propose that generalized transduction can function as a mutualistic trait where temperate phages cooperate with their hosts to survive in rapidly-changing environments. This implies that generalized transduction is not just an error in DNA packaging but is selected for by phages to ensure their survival. [ABSTRACT FROM AUTHOR]

Previous studies have confirmed that acupuncture and moxibustion is an effective way for treating ulcerative colitis (UC). However, the exact mechanism is unclear yet. In this study, DSS-induced UC mice were treated by electroacupuncture and moxibustion, and the genome of intestinal flora was subsequently detected by high-throughput sequencing in order to explore the detailed mechanism in terms of intestinal flora. The results indicated that the alpha diversity indices and beta diversity of intestinal flora were improved by electroacupuncture and moxibustion treatments, especially by the moxibustion treatment. These treatments inhibited Streptococcus, Odoribacter, and Allobaculum whereas it facilitated Lactobacillus on genus level. Further correlation analysis showed that the alpha diversity indices were positively correlated with the percentage of Treg cells in CD4+ cells but negatively correlated with the percentage of Th17 in CD4+ cells. These data indicated that both electroacupuncture and moxibustion can promote the intestinal flora diversity, providing a new view to understand the relationship between host and microbiome when using some external therapies. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of a devastating pig disease present all over the world. The remarkable genetic variation of PRRSV, makes epidemiological and molecular analysis of circulating viruses highly important to review current diagnostic tools and vaccine efficacy. Monitoring PRRS viruses supports modern herd management by explaining the source of found viruses, either internally or externally from the herd. No epidemiological or molecular study has been published on circulating PRRS-viruses in the Netherlands, since the early nineties. Therefore, the objective of this study is to investigate circulating PRRS-viruses in the Netherlands in 2014, 2015 and 2016 on a molecular level by sequencing ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7. The results demonstrate that the 74 PRRSV strains belong to PRRSV-1, but the diversity among strains is high, based on nucleotide identity, individual ORF length and phylogenetic trees of individual ORFs. Furthermore, the data presented here show that the phylogenetic topology of some viruses is ORF dependent and suggests recombination. The identity of the strain of interest might be misinterpreted and wrong conclusions may be drawn in a diagnostic and epidemiological perspective, when only ORF5 is analyzed, as performed in many routine sequencing procedures. [ABSTRACT FROM AUTHOR]

Bloodstream infection (BSI) is a life-threatening condition characterized by the presence of pathogens in the blood. It is associated with increased morbidity and mortality, and has to be treated promptly as mortality increases with every hour of delayed treatment. Therefore, rapid and sensitive diagnosis of BSI is essential. The routine diagnostic method for BSI is blood culture, which can only detect culturable pathogens and takes several days to obtain results. The 16S rRNA gene is present in all bacteria and is commonly used as a target for universal bacterial detection in rapid molecular assays such as PCR. However, molecular detection of the 16S gene is hampered by the large amount of human DNA found in blood samples, making diagnostic results aspecific and less sensitive. We have optimized the selection of PCR primers targeting the 16S rRNA gene to avoid cross-reaction with human DNA background. The developed method increases specificity and sensitivity for pathogen diagnosis, and provides rapid and accurate pathogen detection for rare bacterial DNA in the presence of abundant host DNA. [ABSTRACT FROM AUTHOR]

The development of high-throughput genome sequencing enables accurate measurements of levels of sub-consensus intra-host virus genetic diversity and analysis of the role played by natural selection during cross-species transmission. We analysed the natural and experimental evolution of rabies virus (RABV), an important example of a virus that is able to make multiple host jumps. In particular, we (i) analyzed RABV evolution during experimental host switching with the goal of identifying possible genetic markers of host adaptation, (ii) compared the mutational changes observed during passage with those observed in natura, and (iii) determined whether the colonization of new hosts or tissues requires adaptive evolution in the virus. To address these aims, animal infection models (dog and fox) and primary cell culture models (embryo brain cells of dog and fox) were developed and viral variation was studied in detail through deep genome sequencing. Our analysis revealed a strong unidirectional host evolutionary effect, as dog-adapted rabies virus was able to replicate in fox and fox cells relatively easily, while dogs or neuronal dog cells were not easily susceptible to fox adapted-RABV. This suggests that dog RABV may be able to adapt to some hosts more easily than other host variants, or that when RABV switched from dogs to red foxes it lost its ability to adapt easily to other species. Although no difference in patterns of mutation variation between different host organs was observed, mutations were common following both in vitro and in vivo passage. However, only a small number of these mutations also appeared in natura, suggesting that adaptation during successful cross-species virus transmission is a complex, multifactorial evolutionary process. [ABSTRACT FROM AUTHOR]

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion. [ABSTRACT FROM AUTHOR]

Taxonomy for bacterial isolates is commonly assigned via sequence analysis. However, the most common sequence-based approaches (e.g. 16S rRNA gene-based phylogeny or whole genome comparisons) are still labor intensive and subjective to varying degrees. Here we present a set of 33 bacterial genomes, isolated from the canine oral cavity. Taxonomy of these isolates was first assigned by PCR amplification of the 16S rRNA gene, Sanger sequencing, and taxonomy assignment using BLAST. After genome sequencing, taxonomy was revisited through a manual process using a combination of average nucleotide identity (ANI), concatenated marker gene phylogenies, and 16S rRNA gene phylogenies. This taxonomy was then compared to the automated taxonomic assignment given by the recently proposed Genome Taxonomy Database (GTDB). We found the results of all three methods to be similar (25 out of the 33 had matching genera), but the GTDB approach required fewer subjective decisions, and required far less labor. The primary differences in the non-identical taxonomic assignments involved cases where GTDB has proposed taxonomic revisions. [ABSTRACT FROM AUTHOR]

Precise promoter annotation is required for understanding the mechanistic basis of transcription initiation. In the context of complex genomes, such as herpesviruses where there is extensive genic overlap, identification of transcription start sites (TSSs) is particularly problematic and cannot be comprehensively accessed by standard RNA sequencing approaches. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus and the etiological agent of Kaposi’s sarcoma and the B cell lymphoma primary effusion lymphoma (PEL). Here, we leverage RNA annotation and mapping of promoters for analysis of gene expression (RAMPAGE) and define KSHV TSSs transcriptome-wide and at nucleotide resolution in two widely used models of KSHV infection, namely iSLK.219 cells and the PEL cell line TREx-BCBL1-RTA. By mapping TSSs over a 96 h time course of reactivation we confirm 48 of 50 previously identified TSSs. Moreover, we identify over 100 novel transcription start site clusters (TSCs) in each cell line. Our analyses identified cell-type specific differences in TSC positions as well as promoter strength, and defined motifs within viral core promoters. Collectively, by defining TSSs at high resolution we have greatly expanded the transcriptional landscape of the KSHV genome and identified transcriptional control mechanisms at play during KSHV lytic reactivation. [ABSTRACT FROM AUTHOR]

Virus-host interactions are frequently studied in bulk cell populations, obscuring cell-to-cell variation. Here we investigate endogenous herpesvirus gene expression at the single-cell level, combining a sensitive and robust fluorescent in situ hybridization platform with multiparameter flow cytometry, to study the expression of gammaherpesvirus non-coding RNAs (ncRNAs) during lytic replication, latent infection and reactivation in vitro. This method allowed robust detection of viral ncRNAs of murine gammaherpesvirus 68 (γHV68), Kaposi’s sarcoma associated herpesvirus and Epstein-Barr virus, revealing variable expression at the single-cell level. By quantifying the inter-relationship of viral ncRNA, viral mRNA, viral protein and host mRNA regulation during γHV68 infection, we find heterogeneous and asynchronous gene expression during latency and reactivation, with reactivation from latency identified by a distinct gene expression profile within rare cells. Further, during lytic replication with γHV68, we find many cells have limited viral gene expression, with only a fraction of cells showing robust gene expression, dynamic RNA localization, and progressive infection. Lytic viral gene expression was enhanced in primary fibroblasts and by conditions associated with enhanced viral replication, with multiple subpopulations of cells present in even highly permissive infection conditions. These findings, powered by single-cell analysis integrated with automated clustering algorithms, suggest inefficient or abortive γHV infection in many cells, and identify substantial heterogeneity in viral gene expression at the single-cell level. [ABSTRACT FROM AUTHOR]

Bacteria that are recalcitrant to genetic manipulation using modern in vitro techniques are termed genetically intractable. Genetic intractability is a fundamental barrier to progress that hinders basic, synthetic, and translational microbiology research and development beyond a few model organisms. The most common underlying causes of genetic intractability are restriction-modification (RM) systems, ubiquitous defense mechanisms against xenogeneic DNA that hinder the use of genetic approaches in the vast majority of bacteria and exhibit strain-level variation. Here, we describe a systematic approach to overcome RM systems. Our approach was inspired by a simple hypothesis: if a synthetic piece of DNA lacks the highly specific target recognition motifs for a host's RM systems, then it is invisible to these systems and will not be degraded during artificial transformation. Accordingly, in this process, we determine the genome and methylome of an individual bacterial strain and use this information to define the bacterium's RM target motifs. We then synonymously eliminate RM targets from the nucleotide sequence of a genetic tool in silico, synthesize an RM-silent "SyngenicDNA" tool, and propagate the tool as minicircle plasmids, termed SyMPL (SyngenicDNA Minicircle Plasmid) tools, before transformation. In a proof-of-principle of our approach, we demonstrate a profound improvement (five orders of magnitude) in the transformation of a clinically relevant USA300 strain of Staphylococcus aureus. This stealth-by-engineering SyngenicDNA approach is effective, flexible, and we expect in future applications could enable microbial genetics free of the restraints of restriction-modification barriers. [ABSTRACT FROM AUTHOR]

The article discusses the formation of herpes simplex virus (HSV) replication compartments (RC), as well as their ability to attract cellular and viral proteins and the effect of the interactions between distinct individual RCs. Topics include viral genome amplification, the complex virus-host interactions, and the three stages of HSV-1 viral gene expression, namely, immediate early (IE), early (E), and late (L).

Biochemical process modelling (BPM) is at the process level and driven by observations while cellular level modelling (CLM) is at the cellular level and is driven by knowledge about the genetics and microbial capability. • Multispecies metabolic modelling is essential to analyse anaerobic ecosystems. • Models can be built top down (empirical) or bottom up (based on genome). • Substantial gap in concept and s between the two approaches. • Effective modelling requires principles from both fields. Mixed culture anaerobic processes are important to environmental systems, including the global carbon cycle, and industrial and environmental biotechnology. Mixed culture metabolic modelling (MM) is an essential tool to analyse these systems. MM predicts microbial function based on knowledge or assumption of cellular metabolism. It may be developed based on observations at the process level – biochemical process modelling (BPM) or fundamental knowledge of the cell being modelled – cellular level modelling (CLM). There is a substantial gap between these two fields, with BPM not considering genetic constraints, particularly where this may be important to interspecies interactions (e.g. amino acid transfer), and CLM commonly not considering mass transfer principles, such as advection/diffusion/migration. No unified approach is useful for all applications, but there is an increasing need to consider genetic information and constraints in developing BPM, and translate BPM principles (including mass-transfer and inorganic chemistry) for application to CLM. [ABSTRACT FROM AUTHOR]

Rift Valley fever virus (RVFV) is a negative-sense RNA virus belonging to the Phenuiviridae family that infects both domestic livestock and humans. The NIAID has designated RVFV as a Category A priority emerging pathogen due to the devastating public health outcomes associated with epidemic outbreaks. However, there is no licensed treatment or vaccine approved for human use. Therefore it is of great interest to understand RVFV pathogenesis in infected hosts in order to facilitate creation of targeted therapies and treatment options. Here we provide insight into the host-pathogen interface in human HEK293 cells during RVFV MP-12 strain infection using high-throughput mRNA sequencing technology. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes showed robust innate immune and cytokine-mediated inflammatory pathway activation as well as alterations in pathways associated with fatty acid metabolism and extracellular matrix receptor signaling. We also analyzed the promoter regions of DEGs for patterns in transcription factor binding sites, and found several that are known to act synergistically to impact apoptosis, immunity, metabolism, and cell growth and differentiation. Lastly, we noted dramatic changes in host alternative splicing patterns in genes associated with mRNA decay and surveillance, RNA transport, and DNA repair. This study has improved our understanding of RVFV pathogenesis and has provided novel insight into pathways and signaling modules important for RVFV diagnostics and therapeutic development. [ABSTRACT FROM AUTHOR]