The sheer number of gene variants and the extent of the observed clinical and molecular heterogeneity recorded in neuropsychiatric disorders (NPDs) could be due to the magnified downstream effects initiated by a smaller group of genomic higher-order alterations in response to endogenous or environmental stress. Chromosomal common fragile sites (CFS) are functionally linked with microRNAs, gene copy number variants (CNVs), sub-microscopic deletions and duplications of DNA, rare single-nucleotide variants (SNVs/SNPs), and small insertions/deletions (indels), as well as chromosomal translocations, gene duplications, altered methylation, microRNA and L1 transposon activity, and 3-D chromosomal topology characteristics. These genomic structural features have been linked with various NPDs in mostly isolated reports and have usually only been viewed as areas harboring potential candidate genes of interest. The suggestion to use a higher level entry point (the \'fragilome\' and associated features) activated by a central mechanism (\'stress\') for studying NPD genetics has the potential to unify the existing vast number of different observations in this field. This approach may explain the continuum of gene findings distributed between affected and unaffected individuals, the clustering of NPD phenotypes and overlapping comorbidities, the extensive clinical and molecular heterogeneity, and the association with certain other medical disorders.

Mycoplasma felis has been isolated from diseased cats and horses, but to date only a single fully assembled genome of this species, of an isolate from a horse, has been characterized. This study aimed to characterize and compare the completely assembled genomes of four clinical isolates of M. felis from three domestic cats, assembled with the aid of short- and long-read sequencing methods. The completed genomes encoded a median of 759 ORFs (range 743-777) and had a median average nucleotide identity of 98.2 % with the genome of the available equid origin reference strain. Comparative genomic analysis revealed the occurrence of multiple horizontal gene transfer events and significant genome reassortment. This had resulted in the acquisition or loss of numerous genes within the Australian felid isolate genomes, encoding putative proteins involved in DNA transfer, metabolism, DNA replication, host cell interaction and restriction modification systems. Additionally, a novel mycoplasma phage was detected in one Australian felid M. felis isolate by genomic analysis and visualized using cryo-transmission electron microscopy. This study has highlighted the complex genomic dynamics in different host environments. Furthermore, the sequences obtained in this work will enable the development of new diagnostic tools, and identification of future infection control and treatment options for the respiratory disease complex in cats.

A key step for comparative genomics is to group open reading frames into functionally and evolutionarily meaningful gene clusters. Gene clustering is complicated by intraspecific duplications and horizontal gene transfers that are frequent in prokaryotes. In consequence, gene clustering methods must deal with a trade-off between identifying vertically transmitted representatives of multicopy gene families, which are recognizable by synteny conservation, and retrieving complete sets of species-level orthologs. We studied the implications of adopting homology, orthology, or synteny conservation as formal criteria for gene clustering by performing comparative analyses of 125 prokaryotic pangenomes.
Clustering criteria affect pangenome functional characterization, core genome inference, and reconstruction of ancestral gene content to different extents. Species-wise estimates of pangenome and core genome sizes change by the same factor when using different clustering criteria, allowing robust cross-species comparisons regardless of the clustering criterion. However, cross-species comparisons of genome plasticity and functional profiles are substantially affected by inconsistencies among clustering criteria. Such inconsistencies are driven not only by mobile genetic elements, but also by genes involved in defense, secondary metabolism, and other accessory functions. In some pangenome features, the variability attributed to methodological inconsistencies can even exceed the effect sizes of ecological and phylogenetic variables.
Choosing an appropriate criterion for gene clustering is critical to conduct unbiased pangenome analyses. We provide practical guidelines to choose the right method depending on the research goals and the quality of genome assemblies, and a benchmarking dataset to assess the robustness and reproducibility of future comparative studies.

Halophilic archaea (haloarchaea) are known to exhibit multiple chromosomes, with one main chromosome and one or several smaller secondary chromosomes or megaplasmids. Halorubrum lacusprofundi, a model organism for studying cold adaptation, exhibits one secondary chromosome and one megaplasmid that include a large arsenal of virus defense mechanisms. We isolated a virus (Halorubrum tailed virus DL1, HRTV-DL1) infecting Hrr. lacusprofundi, and present an in-depth characterization of the virus and its interactions with Hrr. lacusprofundi. While studying virus-host interactions between Hrr. lacusprofundi and HRTV-DL1, we uncover that the strain in use (ACAM34_UNSW) lost the entire megaplasmid and about 38% of the secondary chromosome. The loss included the majority of virus defense mechanisms, making the strain sensitive to HRTV-DL1 infection, while the type strain (ACAM34_DSMZ) appears to prevent virus replication. Comparing infection of the type strain ACAM34_DSMZ with infection of the laboratory derived strain ACAM34_UNSW allowed us to identify host responses to virus infection that were only activated in ACAM34_UNSW upon the loss of virus defense mechanisms. We identify one of two S-layer proteins as primary receptor for HRTV-DL1 and conclude that the presence of two different S-layer proteins in one strain provides a strong advantage in the arms race with viruses. Additionally, we identify archaeal homologs to eukaryotic proteins potentially being involved in the defense against virus infection.

Fungal species have dynamic genomes and often exhibit genomic plasticity in response to stress. This genome plasticity often comes with phenotypic consequences that affect fitness and resistance to stress. Fungal pathogens exhibit genome plasticity in both clinical and agricultural settings and often during adaptation to antifungal drugs, posing significant challenges to human health. Therefore, it is important to understand the rates, mechanisms, and impact of large genomic changes. This review addresses the prevalence of polyploidy, aneuploidy, and copy number variation across diverse fungal species, with special attention to prominent fungal pathogens and model species. We also explore the relationship between environmental stress and rates of genomic changes and highlight the mechanisms underlying genotypic and phenotypic changes. A comprehensive understanding of these dynamic fungal genomes is needed to identify novel solutions for the increase in antifungal drug resistance.

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Deinococcus radiopugnans DY59 (formerly Deinococcus swuensis DY59) is a radiation-resistant bacterium isolated from soil. From the 3.5 Mb genomic DNA sequence of strain DY59 (December 2014), 31 insertion sequence (IS) elements of six IS families including IS1, IS4, IS5, IS66, IS630, and IS701 and five unclassified IS elements were detected. Upon induction of oxidative stress with 80 and 100 mM H2O2, the unique ISs of the IS4 family member were actively translocated into a carotenoid biosynthesis gene phytoene desaturase (QR90_10400), resulting in non-pigment phenotypic selection. Therefore, these active transpositions of a specific IS family member were induced by oxidative stress at 80 and 100 mM H2O2. Furthermore, D. radiopugnans DY59 exhibited extremely higher MIC values against H2O2 treatment. To explain this phenomenon, qRT-PCR was conducted to assess the expression levels of catalase and three LysR family regulators. Our findings indicated that the ISDrpg2 and ISDrpg3 elements of the IS4 family were actively transposed into the phytoene desaturase gene by H2O2 treatment via replicative transposition. However, high H2O2 resistance did not originate from H2O2-induced expression of catalase and LysR family regulators.

UNASSIGNED: Free-living amoebae of the Naegleria genus belong to the major protist clade Heterolobosea and are ubiquitously distributed in soil and freshwater habitats. Of the 47 Naegleria species described, N. fowleri is the only one being pathogenic to humans, causing a rare but fulminant primary amoebic meningoencephalitis. Some Naegleria genome sequences are publicly available, but the genetic basis for Naegleria diversity and ability to thrive in diverse environments (including human brain) remains unclear.
UNASSIGNED: Herein, we constructed a high-quality Naegleria genus pangenome to obtain a comprehensive catalog of genes encoded by these amoebae. For this, we first sequenced, assembled, and annotated six new Naegleria genomes.
UNASSIGNED: Genome architecture analyses revealed that Naegleria may use genome plasticity features such as ploidy/aneuploidy to modulate their behavior in different environments. When comparing 14 near-to-complete genome sequences, our results estimated the theoretical Naegleria pangenome as a closed genome, with 13,943 genes, including 3,563 core and 10,380 accessory genes. The functional annotations revealed that a large fraction of Naegleria genes show significant sequence similarity with those already described in other kingdoms, namely Animalia and Plantae. Comparative analyses highlighted a remarkable genomic heterogeneity, even for closely related strains and demonstrate that Naegleria harbors extensive genome variability, reflected in different metabolic repertoires. If Naegleria core genome was enriched in conserved genes essential for metabolic, regulatory and survival processes, the accessory genome revealed the presence of genes involved in stress response, macromolecule modifications, cell signaling and immune response. Commonly reported N. fowleri virulence-associated genes were present in both core and accessory genomes, suggesting that N. fowleri\'s ability to infect human brain could be related to its unique species-specific genes (mostly of unknown function) and/or to differential gene expression. The construction of Naegleria first pangenome allowed us to move away from a single reference genome (that does not necessarily represent each species as a whole) and to identify essential and dispensable genes in Naegleria evolution, diversity and biology, paving the way for further genomic and post-genomic studies.

Leishmania are kinetoplastid pathogens that cause leishmaniasis, a debilitating and potentially life-threatening infection if untreated. Unusually, Leishmania regulate their gene expression largely post-transcriptionally due to the arrangement of their coding genes into polycistronic transcription units that may contain 100s of functionally unrelated genes. Yet, Leishmania are capable of rapid and responsive changes in gene expression to challenging environments, often instead correlating with dynamic changes in their genome composition, ranging from chromosome and gene copy number variations to the generation of extrachromosomal DNA and the accumulation of point mutations. Typically, such events indicate genome instability in other eukaryotes, coinciding with genetic abnormalities, but for Leishmania, exploiting these products of genome instability can provide selectable substrates to catalyse necessary gene expression changes by modifying gene copy number. Unorthodox DNA replication, DNA repair, replication stress factors and DNA repeats are recognised in Leishmania as contributors to this intrinsic instability, but how Leishmania regulate genome plasticity to enhance fitness whilst limiting toxic under- or over-expression of co-amplified and co-transcribed genes is unclear. Herein, we focus on fresh, and detailed insights that improve our understanding of genome plasticity in Leishmania. Furthermore, we discuss emerging models and factors that potentially circumvent regulatory issues arising from polycistronic transcription. Lastly, we highlight key gaps in our understanding of Leishmania genome plasticity and discuss future studies to define, in higher resolution, these complex regulatory interactions.

Heterochromatin is a repressive chromatin state that plays key roles in the functional organisation of eukaryotic genomes. In fungal plant pathogens, effector genes that are required for host colonization tend to be associated with heterochromatic regions of the genome that are enriched with transposable elements. It has been proposed that the heterochromatin environment silences effector genes in the absence of host and dynamic chromatin remodelling facilitates their expression during infection. Here we discuss this model in the context of the key wheat pathogen, Zymoseptoria tritici. We cover progress in understanding the deposition and recognition of heterochromatic histone post translational modifications in Z. tritici and the role that heterochromatin plays in control of genome plasticity and virulence.