Modular Genome Research Articles

Setting Up the JBrowse 2 Genome Browser.

JBrowse 2 is a modular genome browser that can visualize many common genomic file formats. While JBrowse 2 supports a variety of different usages, it is particularly suited for deployment on websites, such as model organism databases or other web-based genomic data resources. This protocol provides detailed instructions for setting up JBrowse 2 on an Ubuntu Linux web server, loading a reference genome from a FASTA format file, and adding a gene annotation track from a GFF3 format file. By the end of the protocol, users will have a working JBrowse 2 instance that is accessible via the web. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Setting up JBrowse 2 on your web server.

Disruption of influenza virus packaging signals results in various misassembled genome complexes.

The influenza A virus genome consists of eight distinct viral RNAs (vRNAs) that are typically packaged into a single virion as an octameric complex. How this genome complex is assembled and incorporated into the virion is poorly understood, but previous research suggests a coordinative role for packaging signals present in all vRNAs. Here, we show that disruption of two packaging signals in a model H7N7 influenza A virus results in a mixture of virions with unusual vRNA content, including empty virions, virions with one to four vRNAs, and virions with octameric complexes composed of vRNA duplicates. Our results suggest that (i) the assembly of error-free octameric complexes proceeds through a series of defined vRNA sub-complexes and (ii) virions can bud without incorporating complete octameric complexes.

JBrowse 2: a modular genome browser with views of synteny and structural variation

We present JBrowse 2, a general-purpose genome annotation browser offering enhanced visualization of complex structural variation and evolutionary relationships. It retains core features of JBrowse while adding new views for synteny, dotplots, breakpoints, gene fusions, and whole-genome overviews. It allows users to share sessions, open multiple genomes, and navigate between views. It can be embedded in a web page, used as a standalone application, or run from Jupyter notebooks or R sessions. These improvements are enabled by a ground-up redesign using modern web technology. We describe application functionality, use cases, performance benchmarks, and implementation notes for web administrators and developers.

A MAD7-based genome editing system for Escherichia coli.

A broad variety of biomolecules is industrially produced in bacteria and yeasts. These microbial expression hosts can be optimized through genetic engineering using CRISPR tools. Here, we designed and characterized such a modular genome editing system based on the Cas12a-like RNA-guided nuclease MAD7 in Escherichia coli. This system enables the efficient generation of single nucleotide polymorphisms (SNPs) or gene deletions and can directly be used with donor DNA from benchtop DNA assembly to increase throughput. We combined multiple edits to engineer an E. coli strain with reduced overflow metabolism and increased plasmid yield, highlighting the versatility and industrial applicability of this approach.

The Clostridium-infecting filamentous phage CAK1 genome analysis allows to define a new potential clade of Tubulavirales.

What we know about Tubulavirales, i.e. filamentous phages, essentially comes from Gram-negative-infecting Inoviridae. However, metagenomics recently suggests filamentous phages are much more widespread and diverse. Here, we report the complete sequence and functional annotation of CAK1, a 6.6kb filamentous phage that was shown to chronically infect Clostridium beijerinckii 30 years ago and only represents the second filamentous phage cultivated on a Gram-positive bacterium. CAK1 has a typical filamentous phage modular genome with no homologs in databases and we were interested to compare it with a pig gut filamentous phage metagenomics dataset that we previously assembled and for which many filamentous phages were predicted to infect Clostridium species by bioinformatics means. CAK1 is distantly related to nine of these sequences, two of which having been predicted as Clostridium-associated. In itself, this small cluster of CAK1-connected sequences sheds light on the diversity of filamentous phages that putatively infect Clostridium species, and probably many other Gram-positive genera.

Co-transfer of functionally interdependent genes contributes to genome mosaicism in lambdoid phages.

Lambdoid (or Lambda-like) phages are a group of related temperate phages that can infect Escherichia coli and other gut bacteria. A key characteristic of these phages is their mosaic genome structure, which served as the basis for the 'modular genome hypothesis'. Accordingly, lambdoid phages evolve by transferring genomic regions, each of which constitutes a functional unit. Nevertheless, it is unknown which genes are preferentially transferred together and what drives such co-transfer events. Here we aim to characterize genome modularity by studying co-transfer of genes among 95 distantly related lambdoid (pro-)phages. Based on gene content, we observed that the genomes cluster into 12 groups, which are characterized by a highly similar gene content within the groups and highly divergent gene content across groups. Highly similar proteins can occur in genomes of different groups, indicating that they have been transferred. About 26 % of homologous protein clusters in the four known operons (i.e. the early left, early right, immunity and late operon) engage in gene transfer, which affects all operons to a similar extent. We identified pairs of genes that are frequently co-transferred and observed that these pairs tend to be near one another on the genome. We find that frequently co-transferred genes are involved in related functions and highlight interesting examples involving structural proteins, the cI repressor and Cro regulator, proteins interacting with DNA, and membrane-interacting proteins. We conclude that epistatic effects, where the functioning of one protein depends on the presence of another, play an important role in the evolution of the modular structure of these genomes.

Highly efficient genome editing in Xanthomonas oryzae pv.oryzae through repurposing the endogenous type I-CCRISPR-Cas system.

Efficient and modular genome editing technologies that manipulate the genome of bacterial pathogens will facilitate the study of pathogenesis mechanisms. However, such methods are yet to be established for Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of rice bacterial blight. We identified a single type I‐C CRISPR‐Cas system in the Xoo genome and leveraged this endogenous defence system for high‐efficiency genome editing in Xoo. Specifically, we developed plasmid components carrying a mini‐CRISPR array, donor DNA, and a phage‐derived recombination system to enable the efficient and programmable genome editing of precise deletions, insertions, base substitutions, and gene replacements. Furthermore, the type I‐C CRISPR‐Cas system of Xoo cleaves target DNA unidirectionally, and this can be harnessed to generate large genomic deletions up to 212 kb efficiently. Therefore, the genome‐editing strategy we have developed can serve as an excellent tool for functional genomics of Xoo, and should also be applicable to other CRISPR‐harbouring bacterial plant pathogens.

A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae.

The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes synthetic neochromosomes as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular neochromosomes of 50 and 100 kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. Neochromosomes made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding neochromosomes were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.

ModHMM: A Modular Supra-Bayesian Genome Segmentation Method.

Genome segmentation methods are powerful tools to obtain cell type or tissue-specific genome-wide annotations and are frequently used to discover regulatory elements. However, traditional segmentation methods show low predictive accuracy and their data-driven annotations have some undesirable properties. As an alternative, we developed ModHMM, a highly modular genome segmentation method. Inspired by the supra-Bayesian approach, it incorporates predictions from a set of classifiers. This allows to compute genome segmentations by utilizing state-of-the-art methodology. We demonstrate the method on ENCODE data and show that it outperforms traditional segmentation methods not only in terms of predictive performance, but also in qualitative aspects. Therefore, ModHMM is a valuable alternative to study the epigenetic and regulatory landscape across and within cell types or tissues.

Chromatin loops associated with active genes and heterochromatin shape rice genome architecture for transcriptional regulation

Insight into high-resolution three-dimensional genome organization and its effect on transcription remains largely elusive in plants. Here, using a long-read ChIA-PET approach, we map H3K4me3- and RNA polymerase II (RNAPII)-associated promoter–promoter interactions and H3K9me2-marked heterochromatin interactions at nucleotide/gene resolution in rice. The chromatin architecture is separated into different independent spatial interacting modules with distinct transcriptional potential and covers approximately 82% of the genome. Compared to inactive modules, active modules possess the majority of active loop genes with higher density and contribute to most of the transcriptional activity in rice. In addition, promoter–promoter interacting genes tend to be transcribed cooperatively. In contrast, the heterochromatin-mediated loops form relative stable structure domains in chromatin configuration. Furthermore, we examine the impact of genetic variation on chromatin interactions and transcription and identify a spatial correlation between the genetic regulation of eQTLs and e-traits. Thus, our results reveal hierarchical and modular 3D genome architecture for transcriptional regulation in rice.

A Negative-Stranded RNA Virus Infecting Citrus Trees: The Second Member of a New Genus Within the Order Bunyavirales.

A new RNA virus has been identified from a sweet orange tree in southern Italy. This virus, tentatively named citrus virus A (CiVA), has a bipartite genome composed of (i) a negative-stranded (ns) RNA1, encoding the viral RNA-dependent RNA polymerase (RdRp), and (ii) an ambisense RNA2, coding for the putative movement protein (MP) and nucleocapsid protein (NP), with the two open reading frames separated by a long AU-rich intergenic region (IR) adopting a hairpin conformation. CiVA genomic RNAs and the encoded proteins resemble those of the recently discovered citrus concave gum-associated virus (CCGaV). This CCGaV, a nsRNA virus associated with the ancient citrus concave gum disease, has been proposed as the representative member of a new genus tentatively named Coguvirus. Molecular and phylogenetic analyses presented here support the classification of CiVA, and likely of other two recently described nsRNA viruses infecting plants, in this new genus. By showing that the evolutionary origin of the MP of all the putative coguviruses likely differs from that of their respective RdRp and NP, this study also provides evidence of a likely modular genome evolution for these viruses. Moreover, phylogenetic data support the proposal that, during the evolutionary history of nsRNA viruses, the plant-infecting viruses most likely emerged from an invertebrate-infecting ancestor several times as independent events. CiVA was identified in a field sweet orange tree not showing any obvious symptom and was graft-transmitted to sweet orange, grapefruit, rough lemon and Dweet tangor indicator plants that did not developed symptoms. The capacity of infecting citrus hosts of several species was also confirmed by a preliminary survey that identified orange, mandarin, clementine and lemon trees as natural hosts of CiVA in several fields of southern Italy, again without any obvious association with specific symptoms.

Reverse Genetics for Newcastle Disease Virus as a Vaccine Vector.

Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and pathogenesis of NDV. Furthermore, NDV has modular genome and accommodates insertion of a foreign gene as a transcriptional unit, thus enabling NDV as a vaccine vector against diseases of humans and animals. Avirulent NDV strains (e.g., LaSota and B1) have been commonly used as vaccine vectors. In this protocol, we have described reverse genetics of NDV to be used as a vaccine vector by exemplifying the recovery of NDV vectored avian influenza virus vaccine. Specifically, cloning and recovery of NDV expressing the hemagglutinin protein of highly pathogenic influenza virus were explained. © 2018 by John Wiley & Sons, Inc.

Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria

BackgroundThere is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions. Another advantage is that mitochondria are suitable organelles to host oxygen sensitive proteins as a low oxygen environment is created within the matrix during cellular respiration.ResultsHere we describe the adaptation of a modular cloning system, GoldenBraid2.0, for the integration of assembled transcriptional units into two different sites of the yeast genome, yielding a high expression level. We have also generated a toolkit comprising various promoters, terminators and selection markers that facilitate the generation of multigenic constructs and allow the reconstruction of biosynthetic pathways within Saccharomyces cerevisiae. To facilitate the specific expression of recombinant proteins within the mitochondrial matrix, we have also included in the toolkit an array of mitochondrial targeting signals and tested their efficiency at different growth conditions. As a proof of concept, we show here the integration and expression of 14 bacterial nitrogen fixation (nif) genes, some of which are known to require specific metallocluster cofactors that contribute to their stability yet make these proteins highly sensitive to oxygen. For one of these genes, nifU, we show that optimal production of this protein is achieved through the use of the Su9 mitochondrial targeting pre-sequence and glycerol as a carbon source to sustain aerobic respiration.ConclusionsWe present here an adapted GoldenBraid2.0 system for modular cloning, genome integration and expression of recombinant proteins in yeast. We have produced a toolkit that includes inducible and constitutive promoters, mitochondrial targeting signals, terminators and selection markers to guarantee versatility in the design of recombinant transcriptional units. By testing the efficiency of the system with nitrogenase Nif proteins and different mitochondrial targeting pre-sequences and growth conditions, we have paved the way for future studies addressing the expression of heterologous proteins in yeast mitochondria.

The first phlebo-like virus infecting plants: a case study on the adaptation of negative-stranded RNA viruses to new hosts.

A novel negative-stranded (ns) RNA virus associated with a severe citrus disease reported more than 80 years ago has been identified. Transmission electron microscopy showed that this novel virus, tentatively named citrus concave gum-associated virus, is flexuous and non-enveloped. Notwithstanding, its two genomic RNAs share structural features with members of the genus Phlebovirus, which are enveloped arthropod-transmitted viruses infecting mammals, and with a group of still unclassified phlebo-like viruses mainly infecting arthropods. CCGaV genomic RNAs code for an RNA-dependent RNA polymerase, a nucleocapsid protein and a putative movement protein showing structural and phylogenetic relationships with phlebo-like viruses, phleboviruses and the unrelated ophioviruses, respectively, thus providing intriguing evidence of a modular genome evolution. Phylogenetic reconstructions identified an invertebrate-restricted virus as the most likely ancestor of this virus, revealing that its adaptation to plants was independent from and possibly predated that of the other nsRNA plant viruses. These data are consistent with an evolutionary scenario in which trans-kingdom adaptation occurred several times during the history of nsRNA viruses and followed different evolutionary pathways, in which genomic RNA segments were gained or lost. The need to create a new genus for this bipartite nsRNA virus and the impact of the rapid and specific detection methods developed here on citrus sanitation and certification are also discussed.

Production of Purified CasRNPs for Efficacious Genome Editing.

CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells. © 2017 by John Wiley & Sons, Inc.

Campylobacter group II phage CP21 is the prototype of a new subgroup revealing a distinct modular genome organization and host specificity

BackgroundThe application of phages is a promising tool to reduce the number of Campylobacter along the food chain. Besides the efficacy against a broad range of strains, phages have to be safe in terms of their genomes. Thus far, no genes with pathogenic potential (e.g., genes encoding virulence factors) have been detected in Campylobacter phages. However, preliminary studies suggested that the genomes of group II phages may be diverse and prone to genomic rearrangements.ResultsWe determined and analysed the genomic sequence (182,761 bp) of group II phage CP21 that is closely related to the already characterized group II phages CP220 and CPt10. The genomes of these phages are comprised of four modules separated by very similar repeat regions, some of which harbouring open reading frames (ORFs). Though, the arrangement of the modules and the location of some ORFs on the genomes are different in CP21 and in CP220/CPt10. In this work, a PCR system was established to study the modular genome organization of other group II phages demonstrating that they belong to different subgroups of the CP220-like virus genus, the prototypes of which are CP21 and CP220. The subgroups revealed different restriction patterns and, interestingly enough, also distinct host specificities, tail fiber proteins and tRNA genes. We additionally analysed the genome of group II phage vB_CcoM-IBB_35 (IBB_35) for which to date only five individual contigs could be determined. We show that the contigs represent modules linked by long repeat regions enclosing some yet not identified ORFs (e.g., for a head completion protein). The data suggest that IBB_35 is a member of the CP220 subgroup.ConclusionCampylobacter group II phages are diverse regarding their genome organization. Since all hitherto characterized group II phages contain numerous genes for transposases and homing endonucleases as well as similar repeat regions, it cannot be excluded that these phages are genetically unstable. To answer this question, further experiments and sequencing of more group II phages should be performed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1837-1) contains supplementary material, which is available to authorized users.

Lysis to Kill: Evaluation of the Lytic Abilities, and Genomics of Nine Bacteriophages Infective for Gordonia spp. and Their Potential Use in Activated Sludge Foam Biocontrol.

Nine bacteriophages (phages) infective for members of the genus Gordonia were isolated from wastewater and other natural water environments using standard enrichment techniques. The majority were broad host range phages targeting more than one Gordonia species. When their genomes were sequenced, they all emerged as double stranded DNA Siphoviridae phages, ranging from 17,562 to 103,424 bp in size, and containing between 27 and 127 genes, many of which were detailed for the first time. Many of these phage genomes diverged from the expected modular genome architecture of other characterized Siphoviridae phages and contained unusual lysis gene arrangements. Whole genome sequencing also revealed that infection with lytic phages does not appear to prevent spontaneous prophage induction in Gordonia malaquae lysogen strain BEN700. TEM sample preparation techniques were developed to view both attachment and replication stages of phage infection.

Three of a Kind: Genetically Similar Tsukamurella Phages TIN2, TIN3, and TIN4.

Three Tsukamurella phages, TIN2, TIN3, and TIN4, were isolated from activated sludge treatment plants located in Victoria, Australia, using conventional enrichment techniques. Illumina and 454 whole-genome sequencing of these Siphoviridae viruses revealed that they had similar genome sequences, ranging in size between 76,268 bp and 76,964 bp. All three phages shared 74% nucleotide sequence identity to the previously described Gordonia phage GTE7. Genome sequencing suggested that phage TIN3 had suffered a mutation in one of its lysis genes compared to the sequence of phage TIN4, to which it is genetically very similar. Mass spectroscopy data showed the unusual presence of a virion structural gene in the DNA replication module of phage TIN4, disrupting the characteristic modular genome architecture of Siphoviridae phages. All three phages appeared highly virulent on strains of Tsukamurella inchonensis and Tsukamurella paurometabola.

Genomic characterization of a temperate phage of the psychrotolerant deep-sea bacterium Aurantimonas sp.

A temperate phage (termed AmM-1) was identified from the psychrotolerant Rhizobiales bacterium Aurantimonas sp. C5-1, which was isolated from bathypelagic water (water depth=1,500m) in the northwest Pacific. The AmM-1 genome is 47,800bp in length and contains 67 coding sequences. Although phage AmM-1 morphologically belongs to the family Myoviridae, its genomic structure, particularly modular genome organization, is similar to that of lambda-type phages of Siphoviridae. Genetic and phylogenetic analyses of the structural core genes also revealed that AmM-1 has a mosaic genomic structure that includes a lambda-like head (Siphoviridae) and P2-like tail (Myoviridae). The sequences of the structural core genes of AmM-1 are distinct from those of previously characterized phage groups but similar to those of recently identified one prophage element and one phage of marine Rhizobiales bacteria: a potential prophage element in the marine psychrotolerant Aureimonas ureilytica DSM 18598 genome and the temperate phage RR-1A infecting Rhizobium radiobacter P007 isolated from deep subseafloor sediment. The mosaic genome structure of AmM-1 suggests the occurrence of genetic exchange between distinct temperate phages in marine Rhizobiales populations.

Genome sequence and characterization of a Rhodococcus equi phage REQ1

Rhodococcus equi is a pathogenic member of the Actinobacteria responsible for causing serious infections in equines. A novel Siphoviridae bacteriophage (REQ1) lytic in R. equi was isolated and characterized. The genome size of REQ1 is 51,342 bp, and its sequence shares 7 % similarity to other DNA sequence in GenBank. Putative open reading frames were identified, and their functions were identified based on their predicted amino acid similarities. REQ1 phage has a modular genome, a feature common in double-stranded DNA phages.