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July 7, 2019

Identification of IncA/C plasmid replication and maintenance genes and development of a plasmid multilocus sequence typing scheme.

Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Genomic changes associated with the evolutionary transition of an insect gut symbiont into a blood-borne pathogen.

The genus Bartonella comprises facultative intracellular bacteria with a unique lifestyle. After transmission by blood-sucking arthropods they colonize the erythrocytes of mammalian hosts causing acute and chronic infectious diseases. Although the pathogen-host interaction is well understood, little is known about the evolutionary origin of the infection strategy manifested by Bartonella species. Here we analyzed six genomes of Bartonella apis, a honey bee gut symbiont that to date represents the closest relative of pathogenic Bartonella species. Comparative genomics revealed that B. apis encodes a large set of vertically inherited genes for amino acid and cofactor biosynthesis and nitrogen metabolism. Most pathogenic bartonellae have lost these ancestral functions, but acquired specific virulence factors and expanded a vertically inherited gene family for harvesting cofactors from the blood. However, the deeply rooted pathogen Bartonella tamiae has retained many of the ancestral genome characteristics reflecting an evolutionary intermediate state toward a host-restricted intraerythrocytic lifestyle. Our findings suggest that the ancestor of the pathogen Bartonella was a gut symbiont of insects and that the adaptation to blood-feeding insects facilitated colonization of the mammalian bloodstream. This study highlights the importance of comparative genomics among pathogens and non-pathogenic relatives to understand disease emergence within an evolutionary-ecological framework.

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July 7, 2019

Geographical structure of endosymbiotic bacteria hosted by Bathymodiolus mussels at eastern Pacific hydrothermal vents.

Chemolithoautotrophic primary production sustains dense invertebrate communities at deep-sea hydrothermal vents and hydrocarbon seeps. Symbiotic bacteria that oxidize dissolved sulfur, methane, and hydrogen gases nourish bathymodiolin mussels that thrive in these environments worldwide. The mussel symbionts are newly acquired in each generation via infection by free-living forms. This study examined geographical subdivision of the thiotrophic endosymbionts hosted by Bathymodiolus mussels living along the eastern Pacific hydrothermal vents. High-throughput sequencing data of 16S ribosomal RNA encoding gene and fragments of six protein-coding genes of symbionts were examined in the samples collected from nine vent localities at the East Pacific Rise, Galápagos Rift, and Pacific-Antarctic Ridge.Both of the parapatric sister-species, B. thermophilus and B. antarcticus, hosted the same numerically dominant phylotype of thiotrophic Gammaproteobacteria. However, sequences from six protein-coding genes revealed highly divergent symbiont lineages living north and south of the Easter Microplate and hosted by these two Bathymodiolus mussel species. High heterogeneity of symbiont haplotypes among host individuals sampled from the same location suggested that stochasticity associated with initial infections was amplified as symbionts proliferated within the host individuals. The mussel species presently contact one another and hybridize along the Easter Microplate, but the northern and southern symbionts appear to be completely isolated. Vicariance associated with orogeny of the Easter Microplate region, 2.5-5.3 million years ago, may have initiated isolation of the symbiont and host populations. Estimates of synonymous substitution rates for the protein-coding bacterial genes examined in this study were 0.77-1.62%/nucleotide/million years.Our present study reports the most comprehensive population genetic analyses of the chemosynthetic endosymbiotic bacteria based on high-throughput genetic data and extensive geographical sampling to date, and demonstrates the role of the geographical features, the Easter Microplate and geographical distance, in the intraspecific divergence of this bacterial species along the mid-ocean ridge axes in the eastern Pacific. Altogether, our results provide insights into extrinsic and intrinsic factors affecting the dispersal and evolution of chemosynthetic symbiotic partners in the hydrothermal vents along the eastern Pacific Ocean.

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July 7, 2019

Coping with living in the soil: the genome of the parthenogenetic springtail Folsomia candida.

Folsomia candida is a model in soil biology, belonging to the family of Isotomidae, subclass Collembola. It reproduces parthenogenetically in the presence of Wolbachia, and exhibits remarkable physiological adaptations to stress. To better understand these features and adaptations to life in the soil, we studied its genome in the context of its parthenogenetic lifestyle.We applied Pacific Bioscience sequencing and assembly to generate a reference genome for F. candida of 221.7 Mbp, comprising only 162 scaffolds. The complete genome of its endosymbiont Wolbachia, was also assembled and turned out to be the largest strain identified so far. Substantial gene family expansions and lineage-specific gene clusters were linked to stress response. A large number of genes (809) were acquired by horizontal gene transfer. A substantial fraction of these genes are involved in lignocellulose degradation. Also, the presence of genes involved in antibiotic biosynthesis was confirmed. Intra-genomic rearrangements of collinear gene clusters were observed, of which 11 were organized as palindromes. The Hox gene cluster of F. candida showed major rearrangements compared to arthropod consensus cluster, resulting in a disorganized cluster.The expansion of stress response gene families suggests that stress defense was important to facilitate colonization of soils. The large number of HGT genes related to lignocellulose degradation could be beneficial to unlock carbohydrate sources in soil, especially those contained in decaying plant and fungal organic matter. Intra- as well as inter-scaffold duplications of gene clusters may be a consequence of its parthenogenetic lifestyle. This high quality genome will be instrumental for evolutionary biologists investigating deep phylogenetic lineages among arthropods and will provide the basis for a more mechanistic understanding in soil ecology and ecotoxicology.

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July 7, 2019

Untangling heteroplasmy, structure, and evolution of an atypical mitochondrial genome by PacBio Sequencing.

The highly compact mitochondrial (mt) genome of terrestrial isopods (Oniscidae) presents two unusual features. First, several loci can individually encode two tRNAs, thanks to single nucleotide polymorphisms at anticodon sites. Within-individual variation (heteroplasmy) at these loci is thought to have been maintained for millions of years because individuals that do not carry all tRNA genes die, resulting in strong balancing selection. Second, the oniscid mtDNA genome comes in two conformations: a ~14 kb linear monomer and a ~28 kb circular dimer comprising two monomer units fused in palindrome. We hypothesized that heteroplasmy actually results from two genome units of the same dimeric molecule carrying different tRNA genes at mirrored loci. This hypothesis, however, contradicts the earlier proposition that dimeric molecules result from the replication of linear monomers-a process that should yield totally identical genome units within a dimer. To solve this contradiction, we used the SMRT (PacBio) technology to sequence mirrored tRNA loci in single dimeric molecules. We show that dimers do present different tRNA genes at mirrored loci; thus covalent linkage, rather than balancing selection, maintains vital variation at anticodons. We also leveraged unique features of the SMRT technology to detect linear monomers closed by hairpins and carrying noncomplementary bases at anticodons. These molecules contain the necessary information to encode two tRNAs at the same locus, and suggest new mechanisms of transition between linear and circular mtDNA. Overall, our analyses clarify the evolution of an atypical mt genome where dimerization counterintuitively enabled further mtDNA compaction. Copyright © 2017 by the Genetics Society of America.

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July 7, 2019

Molecules to ecosystems: Actinomycete natural products in situ.

Actinomycetes, filamentous actinobacteria found in numerous ecosystems around the globe, produce a wide range of clinically useful natural products (NP). In natural environments, actinomycetes live in dynamic communities where environmental cues and ecological interactions likely influence NP biosynthesis. Our current understating of these cues, and the ecological roles of NP, is in its infancy. We postulate that understanding the ecological context in which actinomycete metabolites are made is fundamental to advancing the discovery of novel NP. In this review we explore the ecological relevance of actinomycetes and their secondary metabolites from varying ecosystems, and suggest that investigating the ecology of actinomycete interactions warrants particular attention with respect to metabolite discovery. Furthermore, we focus on the chemical ecology and in situ analysis of actinomycete NP and consider the implications for NP biosynthesis at ecosystem scales.

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July 7, 2019

Discovery of chemoautotrophic symbiosis in the giant shipworm Kuphus polythalamia (Bivalvia: Teredinidae) extends wooden-steps theory.

The “wooden-steps” hypothesis [Distel DL, et al. (2000) Nature 403:725-726] proposed that large chemosynthetic mussels found at deep-sea hydrothermal vents descend from much smaller species associated with sunken wood and other organic deposits, and that the endosymbionts of these progenitors made use of hydrogen sulfide from biogenic sources (e.g., decaying wood) rather than from vent fluids. Here, we show that wood has served not only as a stepping stone between habitats but also as a bridge between heterotrophic and chemoautotrophic symbiosis for the giant mud-boring bivalve Kuphus polythalamia This rare and enigmatic species, which achieves the greatest length of any extant bivalve, is the only described member of the wood-boring bivalve family Teredinidae (shipworms) that burrows in marine sediments rather than wood. We show that K. polythalamia harbors sulfur-oxidizing chemoautotrophic (thioautotrophic) bacteria instead of the cellulolytic symbionts that allow other shipworm species to consume wood as food. The characteristics of its symbionts, its phylogenetic position within Teredinidae, the reduction of its digestive system by comparison with other family members, and the loss of morphological features associated with wood digestion indicate that K. polythalamia is a chemoautotrophic bivalve descended from wood-feeding (xylotrophic) ancestors. This is an example in which a chemoautotrophic endosymbiosis arose by displacement of an ancestral heterotrophic symbiosis and a report of pure culture of a thioautotrophic endosymbiont.

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July 7, 2019

Complete genome sequence of the drought resistance-promoting endophyte Klebsiella sp. LTGPAF-6F.

Bacterial endophytes with capacity to promote plant growth and improve plant tolerance against biotic and abiotic stresses have importance in agricultural practice and phytoremediation. A plant growth-promoting endophyte named Klebsiella sp. LTGPAF-6F, which was isolated from the roots of the desert plant Alhagi sparsifolia in north-west China, exhibits the ability to enhance the growth of wheat under drought stress. The complete genome sequence of this strain consists of one circular chromosome and two circular plasmids. From the genome, we identified genes related to the plant growth promotion and stress tolerance, such as nitrogen fixation, production of indole-3-acetic acid, acetoin, 2,3-butanediol, spermidine and trehalose. This genome sequence provides a basis for understanding the beneficial interactions between LTGPAF-6F and host plants, and will facilitate its applications as biotechnological agents in agriculture. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Comparative genomics of Burkholderia multivorans, a ubiquitous pathogen with a highly conserved genomic structure.

The natural environment serves as a reservoir of opportunistic pathogens. A well-established method for studying the epidemiology of such opportunists is multilocus sequence typing, which in many cases has defined strains predisposed to causing infection. Burkholderia multivorans is an important pathogen in people with cystic fibrosis (CF) and its epidemiology suggests that strains are acquired from non-human sources such as the natural environment. This raises the central question of whether the isolation source (CF or environment) or the multilocus sequence type (ST) of B. multivorans better predicts their genomic content and functionality. We identified four pairs of B. multivorans isolates, representing distinct STs and consisting of one CF and one environmental isolate each. All genomes were sequenced using the PacBio SMRT sequencing technology, which resulted in eight high-quality B. multivorans genome assemblies. The present study demonstrated that the genomic structure of the examined B. multivorans STs is highly conserved and that the B. multivorans genomic lineages are defined by their ST. Orthologous protein families were not uniformly distributed among chromosomes, with core orthologs being enriched on the primary chromosome and ST-specific orthologs being enriched on the second and third chromosome. The ST-specific orthologs were enriched in genes involved in defense mechanisms and secondary metabolism, corroborating the strain-specificity of these virulence characteristics. Finally, the same B. multivorans genomic lineages occur in both CF and environmental samples and on different continents, demonstrating their ubiquity and evolutionary persistence.

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July 7, 2019

Genome analysis of Endomicrobium proavitum suggests loss and gain of relevant functions during the evolution of intracellular symbionts.

Bacterial endosymbionts of eukaryotes show progressive genome erosion, but detailed investigations of the evolutionary processes involved in the transition to an intracellular lifestyle are generally hampered by the lack of extant free-living lineages. Here, we characterize the genome of the recently isolated, free-living Endomicrobium proavitum, the second member of the Elusimicrobia phylum brought into pure culture, and compare it to the closely related “Candidatus Endomicrobium trichonymphae” strain Rs-D17, a previously described but uncultured endosymbiont of termite gut flagellates. A reconstruction of the metabolic pathways of Endomicrobium proavitum matched the fermentation products formed in pure culture and underscored its restriction to glucose as the substrate. However, several pathways present in the free-living strain, e.g., for the uptake and activation of glucose and its subsequent fermentation, ammonium assimilation, and outer membrane biogenesis, were absent or disrupted in the endosymbiont, probably lost during the massive genome rearrangements that occurred during symbiogenesis. While the majority of the genes in strain Rs-D17 have orthologs in Endomicrobium proavitum, the endosymbiont also possesses a number of functions that are absent from the free-living strain and may represent adaptations to the intracellular lifestyle. Phylogenetic analysis revealed that the genes encoding glucose 6-phosphate and amino acid transporters, acetaldehyde/alcohol dehydrogenase, and the pathways of glucuronic acid catabolism and thiamine pyrophosphate biosynthesis were either acquired by horizontal gene transfer or may represent ancestral traits that were lost in the free-living strain. The polyphyletic origin of Endomicrobia in different flagellate hosts makes them excellent models for future studies of convergent and parallel evolution during symbiogenesis.IMPORTANCE The isolation of a free-living relative of intracellular symbionts provides the rare opportunity to identify the evolutionary processes that occur in the course of symbiogenesis. Our study documents that the genome of “Candidatus Endomicrobium trichonymphae,” which represents a clade of endosymbionts that have coevolved with termite gut flagellates for more than 40 million years, is not simply a subset of the genes present in Endomicrobium proavitum, a member of the ancestral, free-living lineage. Rather, comparative genomics revealed that the endosymbionts possess several relevant functions that were either prerequisites for colonization of the intracellular habitat or might have served to compensate for genes losses that occurred during genome erosion. Some gene sets found only in the endosymbiont were apparently acquired by horizontal transfer from other gut bacteria, which suggests that the intracellular bacteria of flagellates are not entirely cut off from gene flow. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Rifamorpholines A-E, potential antibiotics from locust-associated actinobacteria Amycolatopsis sp. Hca4.

Cultivation of locust associated rare actinobacteria, Amycolatopsis sp. HCa4, has provided five unusual macrolactams rifamorpholines A-E. Their structures were determined by interpretation of spectroscopic and crystallographic data. Rifamorpholines A-E possess an unprecedented 5/6/6/6 ring chromophore, representing a new subclass of rifamycin antibiotics. The biosynthetic pathway for compounds 1-5 involves a key 1,6-cyclization for the formation of the morpholine ring. Compounds 2 and 4 showed potent activities against methicillin-resistant Staphylococcus aureus (MRSA) with MICs of 4.0 and 8.0 µM, respectively.

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July 7, 2019

IgA-coated E. coli enriched in Crohn’s disease spondyloarthritis promote TH17-dependent inflammation.

Peripheral spondyloarthritis (SpA) is a common extraintestinal manifestation in patients with active inflammatory bowel disease (IBD) characterized by inflammatory enthesitis, dactylitis, or synovitis of nonaxial joints. However, a mechanistic understanding of the link between intestinal inflammation and SpA has yet to emerge. We evaluated and functionally characterized the fecal microbiome of IBD patients with or without peripheral SpA. Coupling the sorting of immunoglobulin A (IgA)-coated microbiota with 16S ribosomal RNA-based analysis (IgA-seq) revealed a selective enrichment in IgA-coated Escherichia coli in patients with Crohn’s disease-associated SpA (CD-SpA) compared to CD alone. E. coli isolates from CD-SpA-derived IgA-coated bacteria were similar in genotype and phenotype to an adherent-invasive E. coli (AIEC) pathotype. In comparison to non-AIEC E. coli, colonization of germ-free mice with CD-SpA E. coli isolates induced T helper 17 cell (TH17) mucosal immunity, which required the virulence-associated metabolic enzyme propanediol dehydratase (pduC). Modeling the increase in mucosal and systemic TH17 immunity we observed in CD-SpA patients, colonization of interleukin-10-deficient or K/BxN mice with CD-SpA-derived E. coli lead to more severe colitis or inflammatory arthritis, respectively. Collectively, these data reveal the power of IgA-seq to identify immunoreactive resident pathosymbionts that link mucosal and systemic TH17-dependent inflammation and offer microbial and immunophenotype stratification of CD-SpA that may guide medical and biologic therapy. Copyright © 2017, American Association for the Advancement of Science.

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July 7, 2019

Euglena gracilis genome and transcriptome: organelles, nuclear genome assembly strategies and initial features.

Euglena gracilis is a major component of the aquatic ecosystem and together with closely related species, is ubiquitous worldwide. Euglenoids are an important group of protists, possessing a secondarily acquired plastid and are relatives to the Kinetoplastidae, which themselves have global impact as disease agents. To understand the biology of E. gracilis, as well as to provide further insight into the evolution and origins of the Kinetoplastidae, we embarked on sequencing the nuclear genome; the plastid and mitochondrial genomes are already in the public domain. Earlier studies suggested an extensive nuclear DNA content, with likely a high degree of repetitive sequence, together with significant extrachromosomal elements. To produce a list of coding sequences we have combined transcriptome data from both published and new sources, as well as embarked on de novo sequencing using a combination of 454, Illumina paired end libraries and long PacBio reads. Preliminary analysis suggests a surprisingly large genome approaching 2 Gbp, with a highly fragmented architecture and extensive repeat composition. Over 80% of the RNAseq reads from E. gracilis maps to the assembled genome sequence, which is comparable with the well assembled genomes of T. brucei and T. cruzi. In order to achieve this level of assembly we employed multiple informatics pipelines, which are discussed here. Finally, as a preliminary view of the genome architecture, we discuss the tubulin and calmodulin genes, which highlight potential novel splicing mechanisms.

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July 7, 2019

Large scale and significant expression from pseudogenes in Sodalis glossinidius – a facultative bacterial endosymbiont

The majority of bacterial genomes have high coding efficiencies, but there are some genomes of intracellular bacteria that have low gene density. The genome of the endosymbiont Sodalis glossinidius contains almost 50% pseudogenes containing mutations that putatively silence them at the genomic level. We have applied multiple omic strategies, combining: Illumina and Pacific Biosciences Single-Molecule Real Time DNA-sequencing and annotation; stranded RNA-sequencing; and proteome analysis to better understand the transcriptional and translational landscape of Sodalis pseudogenes, and potential mechanisms for their control. Between 53% and 74% of the Sodalis transcriptome remains active in cell-free culture. Mean sense transcription from Coding Domain Sequences (CDS) is four-times greater than that from pseudogenes. Comparative genomic analysis of six Illumina-sequenced Sodalis isolates from different host Glossina species shows pseudogenes make up ~40% of the 2,729 genes in the core genome, suggesting are stable and/or Sodalis is a recent introduction across the Glossina genus as a facultative symbiont. These data further shed light on the importance of transcriptional and translational control in deciphering host-microbe interactions, and demonstrate that pseudogenes are more complex than a simple degrading DNA sequence. The combination of genomics, transcriptomics and proteomics give a multidimensional perspective for studying prokaryotic genomes with a view to elucidating evolutionary adaptation to novel environmental niches.

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July 7, 2019

Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila.

While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the a-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea’s defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.

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