A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.

OMA orthology in 2024: improved prokaryote coverage, ancestral and extant GO enrichment, a revamped synteny viewer and more in the OMA Ecosystem.

In this update paper, we present the latest developments in the OMA browser knowledgebase, which aims to provide high-quality orthology inferences and facilitate the study of gene families, genomes and their evolution. First, we discuss the addition of new species in the database, particularly an expanded representation of prokaryotic species. The OMA browser now offers Ancestral Genome pages and an Ancestral Gene Order viewer, allowing users to explore the evolutionary history and gene content of ancestral genomes. We also introduce a revamped Local Synteny Viewer to compare genomic neighborhoods across both extant and ancestral genomes. Hierarchical Orthologous Groups (HOGs) are now annotated with Gene Ontology annotations, and users can easily perform extant or ancestral GO enrichments. Finally, we recap new tools in the OMA Ecosystem, including OMAmer for proteome mapping, OMArk for proteome quality assessment, OMAMO for model organism selection and Read2Tree for phylogenetic species tree construction from reads. These new features provide exciting opportunities for orthology analysis and comparative genomics. OMA is accessible at https://omabrowser.org.

RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila.

How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.

Mitochondrial Genome Evolution in Annelida-A Systematic Study on Conservative and Variable Gene Orders and the Factors Influencing its Evolution.

The mitochondrial genomes of Bilateria are relatively conserved in their protein-coding, rRNA, and tRNA gene complement, but the order of these genes can range from very conserved to very variable depending on the taxon. The supposedly conserved gene order of Annelida has been used to support the placement of some taxa within Annelida. Recently, authors have cast doubts on the conserved nature of the annelid gene order. Various factors may influence gene order variability including, among others, increased substitution rates, base composition differences, structure of noncoding regions, parasitism, living in extreme habitats, short generation times, and biomineralization. However, these analyses were neither done systematically nor based on well-established reference trees. Several focused on only a few of these factors and biological factors were usually explored ad-hoc without rigorous testing or correlation analyses. Herein, we investigated the variability and evolution of the annelid gene order and the factors that potentially influenced its evolution, using a comprehensive and systematic approach. The analyses were based on 170 genomes, including 33 previously unrepresented species. Our analyses included 706 different molecular properties, 20 life-history and ecological traits, and a reference tree corresponding to recent improvements concerning the annelid tree. The results showed that the gene order with and without tRNAs is generally conserved. However, individual taxa exhibit higher degrees of variability. None of the analyzed life-history and ecological traits explained the observed variability across mitochondrial gene orders. In contrast, the combination and interaction of the best-predicting factors for substitution rate and base composition explained up to 30% of the observed variability. Accordingly, correlation analyses of different molecular properties of the mitochondrial genomes showed an intricate network of direct and indirect correlations between the different molecular factors. Hence, gene order evolution seems to be driven by molecular evolutionary aspects rather than by life history or ecology. On the other hand, variability of the gene order does not predict if a taxon is difficult to place in molecular phylogenetic reconstructions using sequence data or not. We also discuss the molecular properties of annelid mitochondrial genomes considering canonical views on gene evolution and potential reasons why the canonical views do not always fit to the observed patterns without making some adjustments. [Annelida; compositional biases; ecology; gene order; life history; macroevolution; mitochondrial genomes; substitution rates.].

Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure.

Paramutation is a form of non-Mendelian inheritance in which the expression of a paramutable allele changes when it encounters a paramutagenic allele. This change in expression of the paramutable alleles is stably inherited even after segregation of both alleles. While the discovery of paramutation and studies of its underlying mechanism were made with alleles that change plant pigmentation, paramutation-like phenomena are known to modulate the expression of other traits and in other eukaryotes, and many cases have probably gone undetected. It is likely that epigenetic mechanisms are responsible for the phenomenon, as paramutation forms epialleles, genes with identical sequences but different expression states. This could account for the intergenerational inheritance of the paramutated allele, providing profound evidence that triggered epigenetic changes can be maintained over generations. Here, we use a case of paramutation that affects a transgenic selection reporter gene in tetraploid Arabidopsis thaliana. Our data suggest that different types of small RNA are derived from paramutable and paramutagenic epialleles. In addition, deletion of a repeat within the epiallele changes its paramutability. Further, the temperature during the growth of the epiallelic hybrids determines the degree and timing of the allelic interaction. The data further make it plausible why paramutation in this system becomes evident only in the segregating F2 population of tetraploid plants containing both epialleles. In summary, the results support a model for polyploidy-associated paramutation, with similarities as well as distinctions from other cases of paramutation.

Doublesex regulates fruitless expression to promote sexual dimorphism of the gonad stem cell niche.

Doublesex (Dsx) and Fruitless (Fru) are the two downstream transcription factors that actuate Drosophila sex determination. While Dsx assists Fru to regulate sex-specific behavior, whether Fru collaborates with Dsx in regulating other aspects of sexual dimorphism remains unknown. One important aspect of sexual dimorphism is found in the gonad stem cell (GSC) niches, where male and female GSCs are regulated to create large numbers of sperm and eggs. Here we report that Fru is expressed male-specifically in the GSC niche and plays important roles in the development and maintenance of these cells. Unlike previously-studied aspects of sex-specific Fru expression, which are regulated by Transformer (Tra)-mediated alternative splicing, we show that male-specific expression of fru in the gonad is regulated downstream of dsx, and is independent of tra. fru genetically interacts with dsx to support maintenance of the niche throughout development. Ectopic expression of fru inhibited female niche formation and partially masculinized the ovary. fru is also required autonomously for cyst stem cell maintenance and cyst cell survival. Finally, we identified a conserved Dsx binding site upstream of fru promoter P4 that regulates fru expression in the niche, indicating that fru is likely a direct target for transcriptional regulation by Dsx. These findings demonstrate that fru acts outside the nervous system to influence sexual dimorphism and reveal a new mechanism for regulating sex-specific expression of fru that is regulated at the transcriptional level by Dsx, rather than by alternative splicing by Tra.

NCK-associated protein 1 like (nckap1l) minor splice variant regulates intrahepatic biliary network morphogenesis.

Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.

Complete Mitochondrial Genomes and Phylogenetic Analysis of Genus Henricia (Asteroidea: Spinulosida: Echinasteridae).

The genus Henricia is known to have intraspecific morphological variations, making species identification difficult. Therefore, molecular phylogeny analysis based on genetic characteristics is valuable for species identification. We present complete mitochondrial genomic sequences of Henricia longispina aleutica, H. reniossa, and H. sanguinolenta for the first time in this study. This study will make a significant contribution to our understanding of Henricia species and its relationships within the class Asteroidea. Lengths of mitochondrial genomes of the three species are 16,217, 16,223, and 16,194 bp, respectively, with a circular form. These genomes contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a D-loop. The gene order and direction aligned with other asteroid species. Phylogenetic relationship analysis showed that our Henricia species were in a monophyletic clade with other Henricia species and in a large clade with species (Echinaster brasiliensis) from the same family. These findings provide valuable insight into understanding the phylogenetic relationships of species in the genus Henricia.

Positive Selection during Niche Adaptation Results in Large-Scale and Irreversible Rearrangement of Chromosomal Gene Order in Bacteria.

Analysis of bacterial genomes shows that, whereas diverse species share many genes in common, their linear order on the chromosome is often not conserved. Whereas rearrangements in gene order could occur by genetic drift, an alternative hypothesis is rearrangement driven by positive selection during niche adaptation (SNAP). Here, we provide the first experimental support for the SNAP hypothesis. We evolved Salmonella to adapt to growth on malate as the sole carbon source and followed the evolutionary trajectories. The initial adaptation to growth in the new environment involved the duplication of 1.66 Mb, corresponding to one-third of the Salmonella chromosome. This duplication is selected to increase the copy number of a single gene, dctA, involved in the uptake of malate. Continuing selection led to the rapid loss or mutation of duplicate genes from either copy of the duplicated region. After 2000 generations, only 31% of the originally duplicated genes remained intact and the gene order within the Salmonella chromosome has been significantly and irreversibly altered. These results experientially validate predictions made by the SNAP hypothesis and show that SNAP can be a strong driving force for rearrangements in chromosomal gene order.

The complete mitochondrial genome and novel gene arrangement in Nesodiprion zhejiangensis Zhou & Xiao (Hymenoptera: Diprionidae).

The complete mitochondrial genome (mitogenome) of the sawfly, Nesodiprion zhejiangensis Zhou & Xiao, was sequenced, assembled, and deposited in GenBank (Accession Number: OM501121). The 15,660 bp N. zhejiangensis mitogenome encodes for 2 ribosomal RNAs (rrnL and rrnS), 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), and an AT-rich region of 450 bp in length. The nucleotide composition is biased toward adenine and thymine (A + T = 81.8%). Each PCG is initiated by an ATN codon, except for cox2, which starts with a TTG. Of 13 PCGs, 9 have a TAA termination codon, while the remainder terminate with a TAG or a single T. All tRNAs have the classic cloverleaf structure, except for the dihydrouridine (DHU) arm of tRNAval, which forms a simple loop. There are 49 helices belonging to 6 domains in rrnL and 30 helices belonging to 4 domains in rrnS. In comparison to the ancestral architecture, N. zhejiangensis has the most rearranged mitogenome in Symphyta, in which rearrangement events of local inversion and transposition are identified in three gene clusters. Specifically, the main hotspot of gene rearrangement occurred between rrnS and trnY, and rearranged from rrnS-(AT-rich region)-I-Q-M-nd2-W-C-Y to rrnS-Q-W-C-nd2-I-M-(AT-rich region)-Y, involving a local inversion event of a large gene cluster and transposition events of some tRNAs. Transposition of trnA and trnR (rearranged from A-R to R-A) was observed at the nd3-nd5 gene junction while shuffling of trnP and trnT (rearranged from T-P to P-T) occurred at the nd4l-nd6 gene junction. While illegitimate inter-mtDNA recombination might explain the opposite orientations of transcription between rrnS and trnY, transposition events of tRNA in some gene blocks can be accounted for by the tandem duplication/random loss (TDRL) model. Our phylogenetic analysis suggests that N. zhejiangensis is closely related to congeneric species N. biremis and N. japonicus, which together form a sister lineage with the European pine sawfly, Neodiprion sertifer.

Gene Positioning Effects on Expression in Eukaryotes.

The packaging and organization of the genome within the eukaryotic interphase nucleus directly influence how the genes are expressed. An underappreciated aspect of genome structure is that it is highly dynamic and that the physical positioning of a gene can impart control over its transcriptional status. In this review, we assess the current knowledge of how gene positioning at different levels of genome organization can directly influence gene expression during interphase. The levels of organization discussed include chromatin looping, topologically associated domains, chromosome territories, and nuclear compartments. We discuss specific studies demonstrating that gene positioning is a dynamic and highly regulated feature of the eukaryotic genome that allows for the essential spatiotemporal regulation of genes.

Powassan Viruses Spread Cell to Cell during Direct Isolation from Ixodes Ticks and Persistently Infect Human Brain Endothelial Cells and Pericytes.

Powassan viruses (POWVs) are neurovirulent tick-borne flaviviruses emerging in the northeastern United States, with a 2% prevalence in Long Island (LI) deer ticks (Ixodes scapularis). POWVs are transmitted within as little as 15 min of a tick bite and enter the central nervous system (CNS) to cause encephalitis (10% of cases are fatal) and long-term neuronal damage. POWV-LI9 and POWV-LI41 present in LI Ixodes ticks were isolated by directly inoculating VeroE6 cells with tick homogenates and detecting POWV-infected cells by immunoperoxidase staining. Inoculated POWV-LI9 and LI41 were exclusively present in infected cell foci, indicative of cell to cell spread, despite growth in liquid culture without an overlay. Cloning and sequencing establish POWV-LI9 as a phylogenetically distinct lineage II POWV strain circulating in LI deer ticks. Primary human brain microvascular endothelial cells (hBMECs) and pericytes form a neurovascular complex that restricts entry into the CNS. We found that POWV-LI9 and -LI41 and lineage I POWV-LB productively infect hBMECs and pericytes and that POWVs were basolaterally transmitted from hBMECs to lower-chamber pericytes without permeabilizing polarized hBMECs. Synchronous POWV-LI9 infection of hBMECs and pericytes induced proinflammatory chemokines, interferon-ß (IFN-ß) and proteins of the IFN-stimulated gene family (ISGs), with delayed IFN-ß secretion by infected pericytes. IFN inhibited POWV infection, but despite IFN secretion, a subset of POWV-infected hBMECs and pericytes remained persistently infected. These findings suggest a potential mechanism for POWVs (LI9/LI41 and LB) to infect hBMECs, spread basolaterally to pericytes, and enter the CNS. hBMEC and pericyte responses to POWV infection suggest a role for immunopathology in POWV neurovirulence and potential therapeutic targets for preventing POWV spread to neuronal compartments. IMPORTANCE We isolated POWVs from LI deer ticks (I. scapularis) directly in VeroE6 cells, and sequencing revealed POWV-LI9 as a distinct lineage II POWV strain. Remarkably, inoculation of VeroE6 cells with POWV-containing tick homogenates resulted in infected cell foci in liquid culture, consistent with cell-to-cell spread. POWV-LI9 and -LI41 and lineage I POWV-LB strains infected hBMECs and pericytes that comprise neurovascular complexes. POWVs were nonlytically transmitted basolaterally from infected hBMECs to lower-chamber pericytes, suggesting a mechanism for POWV transmission across the blood-brain barrier (BBB). POWV-LI9 elicited inflammatory responses from infected hBMEC and pericytes that may contribute to immune cell recruitment and neuropathogenesis. This study reveals a potential mechanism for POWVs to enter the CNS by infecting hBMECs and spreading basolaterally to abluminal pericytes. Our findings reveal that POWV-LI9 persists in cells that form a neurovascular complex spanning the BBB and suggest potential therapeutic targets for preventing POWV spread to neuronal compartments.

Evolution of mitochondrial and nuclear genomes in Pennatulacea.

We examine the phylogeny of sea pens using sequences of whole mitochondrial genomes and the nuclear ribosomal cluster generated through low coverage Illumina sequencing. Taxon sampling includes 30 species in 19 genera representing 13 families. Ancestral state reconstruction shows that most sea pen mitochondrial genomes have the ancestral gene order, and that Pennatulacea with diverse gene orders are found in a single clade. The monophyly of Pennatulidae and Protoptilidae are rejected by both the mitochondrial and nuclear dataset, while the mitochondrial dataset further rejects monophyly of Virgulariidae, and the nuclear dataset rejects monophyly of Kophobelemnidae. We show discordance between nuclear ribosomal gene cluster phylogenies and whole mitochondrial genome phylogenies and highlight key Pennatulacea taxa that could be included in cnidarian genome-wide studies to better resolve the sea pen tree of life. We further illustrate how well frequently sequenced markers capture the overall diversity of the mitochondrial genome and the nuclear ribosomal genes in sea pens.

Evolution of mitogenomic gene order in Orthoptera.

Mitochondrial gene order has contributed to the elucidation of evolutionary relationships in several animal groups. It generally has found its application as a phylogenetic marker for deep nodes. Yet, in Orthoptera limited research has been performed on the gene order, although the group represents one of the oldest insect orders. We performed a comprehensive study on mitochondrial genome rearrangements (MTRs) within Orthoptera in the context of mitogenomic sequence-based phylogeny. We used 280 published mitogenome sequences from 256 species, including three outgroup species, to reconstruct a molecular phylogeny. Using a heuristic approach, we assigned MTR scenarios to the edges of the phylogenetic tree and reconstructed ancestral gene orders to identify possible synapomorphies in Orthoptera. We found all types of MTRs in our dataset: inversions, transpositions, inverse transpositions, and tandem-duplication/random loss events (TDRL). Most of the suggested MTRs were in single and unrelated species. Out of five MTRs which were unique in subgroups of Orthoptera, we suggest four of them to be synapomorphies; those were in the infraorder Acrididea, in the tribe Holochlorini, in the subfamily Pseudophyllinae, and in the two families Phalangopsidae and Gryllidae or their common ancestor (leading to the relationship ((Phalangopsidae + Gryllidae) + Trigonidiidae)). However, similar MTRs have been found in distant insect lineages. Our findings suggest convergent evolution of specific mitochondrial gene orders in several species, deviant from the evolution of the mitogenome DNA sequence. As most MTRs were detected at terminal nodes, a phylogenetic inference of deeper nodes based on MTRs is not supported. Hence, the marker does not seem to aid resolving the phylogeny of Orthoptera, but adds further evidence for the complex evolution of the whole group, especially at the genetic and genomic levels. The results indicate a high demand for more research on patterns and underlying mechanisms of MTR events in Orthoptera.

OMA orthology in 2021: website overhaul, conserved isoforms, ancestral gene order and more.

OMA is an established resource to elucidate evolutionary relationships among genes from currently 2326 genomes covering all domains of life. OMA provides pairwise and groupwise orthologs, functional annotations, local and global gene order conservation (synteny) information, among many other functions. This update paper describes the reorganisation of the database into gene-, group- and genome-centric pages. Other new and improved features are detailed, such as reporting of the evolutionarily best conserved isoforms of alternatively spliced genes, the inferred local order of ancestral genes, phylogenetic profiling, better cross-references, fast genome mapping, semantic data sharing via RDF, as well as a special coronavirus OMA with 119 viruses from the Nidovirales order, including SARS-CoV-2, the agent of the COVID-19 pandemic. We conclude with improvements to the documentation of the resource through primers, tutorials and short videos. OMA is accessible at https://omabrowser.org.

The SNAP hypothesis: Chromosomal rearrangements could emerge from positive Selection during Niche Adaptation.

The relative linear order of most genes on bacterial chromosomes is not conserved over evolutionary timescales. One explanation is that selection is weak, allowing recombination to randomize gene order by genetic drift. However, most chromosomal rearrangements are deleterious to fitness. In contrast, we propose the hypothesis that rearrangements in gene order are more likely the result of selection during niche adaptation (SNAP). Partial chromosomal duplications occur very frequently by recombination between direct repeat sequences. Duplicated regions may contain tens to hundreds of genes and segregate quickly unless maintained by selection. Bacteria exposed to non-lethal selections (for example, a requirement to grow on a poor nutrient) can adapt by maintaining a duplication that includes a gene that improves relative fitness. Further improvements in fitness result from the loss or inactivation of non-selected genes within each copy of the duplication. When genes that are essential in single copy are lost from different copies of the duplication, segregation is prevented even if the original selection is lifted. Functional gene loss continues until a new genetic equilibrium is reached. The outcome is a rearranged gene order. Mathematical modelling shows that this process of positive selection to adapt to a new niche can rapidly drive rearrangements in gene order to fixation. Signature features (duplication formation and divergence) of the SNAP model were identified in natural isolates from multiple species showing that the initial two steps in the SNAP process can occur with a remarkably high frequency. Further bioinformatic and experimental analyses are required to test if and to which extend the SNAP process acts on bacterial genomes.

Fragmentation in mitochondrial genomes in relation to elevated sequence divergence and extreme rearrangements.

BACKGROUND: A single circular mitochondrial (mt) genome is a common feature across most metazoans. The mt-genome includes protein-coding genes involved in oxidative phosphorylation, as well as RNAs necessary for translation of mt-RNAs, whose order and number are highly conserved across animal clades, with few known exceptions of alternative mt-gene order or mt-genome architectures. One such exception consists of the fragmented mitochondrial genome, a type of genome architecture where mt-genes are split across two or more mt-chromosomes. However, the origins of mt-genome fragmentation and its effects on mt-genome evolution are unknown. Here, we investigate these origin and potential mechanisms underlying mt-genome fragmentation, focusing on a genus of booklice, Liposcelis, which exhibits elevated sequence divergence, frequent rearrangement of mt-gene order, and fragmentation of the mt genome, and compare them to other Metazoan clades. RESULTS: We found this genus Liposcelis exhibits very low conservation of mt-gene order across species, relative to other metazoans. Levels of gene order rearrangement were, however, unrelated to whether or not mt-genomes were fragmented or intact, suggesting mitochondrial genome fragmentation is not affecting mt-gene order directly. We further investigated possible mechanisms underpinning these patterns and revealed very high conservation of non-coding sequences at the edges of multiple recombination regions across populations of one particular Liposcelis species, supportive of a hypothesis that mt-fragmentation arises from recombination errors between mt-genome copies. We propose these errors may arise as a consequence of a heightened mutation rate in clades exhibiting mt-fragmentation. Consistent with this, we observed a striking pattern across three Metazoan phyla (Arthropoda, Nematoda, Cnidaria) characterised by members exhibiting high levels of mt-gene order rearrangement and cases of mt-fragmentation, whereby the mt-genomes of species more closely related to species with fragmented mt-genomes diverge more rapidly despite experiencing strong purifying selection. CONCLUSIONS: We showed that contrary to expectations, mt-genome fragmentation is not correlated with the increase in mt-genome rearrangements. Furthermore, we present evidence that fragmentation of the mt-genome may be part of a general relaxation of a natural selection on the mt-genome, thus providing new insights into the origins of mt-genome fragmentation and evolution.

Analysis of Six tonB Gene Homologs in Bacteroides fragilis Revealed That tonB3 is Essential for Survival in Experimental Intestinal Colonization and Intra-Abdominal Infection.

The opportunistic, anaerobic pathogen and commensal of the human large intestinal tract, Bacteroides fragilis strain 638R, contains six predicted TonB proteins, termed TonB1-6, four ExbBs orthologs, ExbB1-4, and five ExbDs orthologs, ExbD1-5. The inner membrane TonB/ExbB/ExbD complex harvests energy from the proton motive force (Δp), and the TonB C-terminal domain interacts with and transduces energy to outer membrane TonB-dependent transporters (TBDTs). However, TonB's role in activating nearly one hundred TBDTs for nutrient acquisition in B. fragilis during intestinal colonization and extraintestinal infection has not been established. In this study, we show that growth was abolished in the ΔtonB3 mutant when heme, vitamin B12, Fe(III)-ferrichrome, starch, mucin-glycans, or N-linked glycans were used as a substrate for growth in vitro. Genetic complementation of the ΔtonB3 mutant with the tonB3 gene restored growth on these substrates. The ΔtonB1, ΔtonB2, ΔtonB4, ΔtonB5, and ΔtonB6 single mutants did not show a growth defect. This indicates that there was no functional compensation for the lack of TonB3, and it demonstrates that TonB3, alone, drives the TBDTs involved in the transport of essential nutrients. The ΔtonB3 mutant had a severe growth defect in a mouse model of intestinal colonization compared to the parent strain. This intestinal growth defect was enhanced in the ΔtonB3 ΔtonB6 double mutant strain, which completely lost its ability to colonize the mouse intestinal tract compared to the parent strain. The ΔtonB1, ΔtonB2, ΔtonB4, and ΔtonB5 mutants did not significantly affect intestinal colonization. Moreover, the survival of the ΔtonB3 mutant strain was completely eradicated in a rat model of intra-abdominal infection. Taken together, these findings show that TonB3 was essential for survival in vivo. The genetic organization of tonB1, tonB2, tonB4, tonB5, and tonB6 gene orthologs indicates that they may interact with periplasmic and nonreceptor outer membrane proteins, but the physiological relevance of this has not been defined. Because anaerobic fermentation metabolism yields a lower Δp than aerobic respiration and B. fragilis has a reduced redox state in its periplasmic space-in contrast to an oxidative environment in aerobes-it remains to be determined if the diverse system of TonB/ExbB/ExbD orthologs encoded by B. fragilis have an increased sensitivity to PMF (relative to aerobic bacteria) to allow for the harvesting of energy under anaerobic conditions.

Emergence of distinct syntenic density regimes is associated with early metazoan genomic transitions.

BACKGROUND: Animal genomes are strikingly conserved in terms of local gene order (microsynteny). While some of these microsyntenies have been shown to be coregulated or to form gene regulatory blocks, the diversity of their genomic and regulatory properties across the metazoan tree of life remains largely unknown. RESULTS: Our comparative analyses of 49 animal genomes reveal that the largest gains of synteny occurred in the last common ancestor of bilaterians and cnidarians and in that of bilaterians. Depending on their node of emergence, we further show that novel syntenic blocks are characterized by distinct functional compositions (Gene Ontology terms enrichment) and gene density properties, such as high, average and low gene density regimes. This is particularly pronounced among bilaterian novel microsyntenies, most of which fall into high gene density regime associated with higher gene coexpression levels. Conversely, a majority of vertebrate novel microsyntenies display a low gene density regime associated with lower gene coexpression levels. CONCLUSIONS: Our study provides first evidence for evolutionary transitions between different modes of microsyntenic block regulation that coincide with key events of metazoan evolution. Moreover, the microsyntenic profiling strategy and interactive online application (Syntenic Density Browser, available at: http://synteny.csb.univie.ac.at/ ) we present here can be used to explore regulatory properties of microsyntenic blocks and predict their coexpression in a wide-range of animal genomes.

Phylogenetic position of Bopyroides hippolytes, with comments on the rearrangement of the mitochondrial genome in isopods (Isopoda: Epicaridea: Bopyridae).

BACKGROUND: Classification of parasitic bopyrids has traditionally been based on morphological characteristics, but phylogenetic relationships have remained elusive due to limited information provided by morphological data and tendency for loss of morphological features as a result of parasitic lifestyle. Subfamily Argeiinae was separated from Bopyrinae based on morphological evidence, although the assignment of all genera has not been phylogenetically evaluated. Bopyroides hippolytes has been traditionally classified in Bopyrinae, but divergent morphological characters make this assignment questionable. To investigate the relationship of bopyrines, we sequenced the complete mitochondrial genome of B. hippolytes and four mitochondrial genes of two other Bopyrinae. RESULTS: The phylogenetic trees based on separate and combined cox1and 18S sequence data recovered Bopyridae as robustly monophyletic, but Bopyrinae as polyphyletic. Bopyroides hippolytes was a close sister to Argeia pugettensis, type species to Argeiinae. Mitochondrial phylogenomics also suggested that B. hippolytes was close to Argeiinae. We also found a novel gene order in B. hippolytes compared to other isopods. CONCLUSIONS: Bopyroides hippolytes should be excluded from the Bopyrinae and has a close affinity with Argeia pugettensis based on molecular and morphological data. The conserved syntenic blocks of mitochondrial gene order have distinctive characteristics at a subordinal level and may be helpful for understanding the higher taxonomic level relationships of Isopoda.