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July 7, 2019

Assembly and transfer of tripartite integrative and conjugative genetic elements.

Integrative and conjugative elements (ICEs) are ubiquitous mobile genetic elements present as “genomic islands” within bacterial chromosomes. Symbiosis islands are ICEs that convert nonsymbiotic mesorhizobia into symbionts of legumes. Here we report the discovery of symbiosis ICEs that exist as three separate chromosomal regions when integrated in their hosts, but through recombination assemble as a single circular ICE for conjugative transfer. Whole-genome comparisons revealed exconjugants derived from nonsymbiotic mesorhizobia received three separate chromosomal regions from the donor Mesorhizobium ciceri WSM1271. The three regions were each bordered by two nonhomologous integrase attachment (att) sites, which together comprised three homologous pairs of attL and attR sites. Sequential recombination between each attL and attR pair produced corresponding attP and attB sites and joined the three fragments to produce a single circular ICE, ICEMcSym(1271) A plasmid carrying the three attP sites was used to recreate the process of tripartite ICE integration and to confirm the role of integrase genes intS, intM, and intG in this process. Nine additional tripartite ICEs were identified in diverse mesorhizobia and transfer was demonstrated for three of them. The transfer of tripartite ICEs to nonsymbiotic mesorhizobia explains the evolution of competitive but suboptimal N2-fixing strains found in Western Australian soils. The unheralded existence of tripartite ICEs raises the possibility that multipartite elements reside in other organisms, but have been overlooked because of their unusual biology. These discoveries reveal mechanisms by which integrases dramatically manipulate bacterial genomes to allow cotransfer of disparate chromosomal regions.


July 7, 2019

Selvamicin, an atypical antifungal polyene from two alternative genomic contexts.

The bacteria harbored by fungus-growing ants produce a variety of small molecules that help maintain a complex multilateral symbiosis. In a survey of antifungal compounds from these bacteria, we discovered selvamicin, an unusual antifungal polyene macrolide, in bacterial isolates from two neighboring ant nests. Selvamicin resembles the clinically important antifungals nystatin A1 and amphotericin B, but it has several distinctive structural features: a noncationic 6-deoxymannose sugar at the canonical glycosylation site and a second sugar, an unusual 4-O-methyldigitoxose, at the opposite end of selvamicin’s shortened polyene macrolide. It also lacks some of the pharmacokinetic liabilities of the clinical agents and appears to have a different target. Whole genome sequencing revealed the putative type I polyketide gene cluster responsible for selvamicin’s biosynthesis including a subcluster of genes consistent with selvamicin’s 4-O-methyldigitoxose sugar. Although the selvamicin biosynthetic cluster is virtually identical in both bacterial producers, in one it is on the chromosome, in the other it is on a plasmid. These alternative genomic contexts illustrate the biosynthetic gene cluster mobility that underlies the diversity and distribution of chemical defenses by the specialized bacteria in this multilateral symbiosis.


July 7, 2019

The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II)2Cys6 transcription factor BcBot6.

Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G+C/A+T-equilibrated regions that contain the Bcbot genes alternate with A+T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)2Cys6 putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the ?Bcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BOT synthesis and of its ecological role in the B. cinerea life cycle. Copyright © 2016 Elsevier Inc. All rights reserved.


July 7, 2019

Complete genome sequence and transcriptomic analysis of a novel marine strain Bacillus weihaiensis reveals the mechanism of brown algae degradation.

A novel marine strain representing efficient degradation ability toward brown algae was isolated, identified, and assigned to Bacillus weihaiensis Alg07. The alga-associated marine bacteria promote the nutrient cycle and perform important functions in the marine ecosystem. The de novo sequencing of the B. weihaiensis Alg07 genome was carried out. Results of gene annotation and carbohydrate-active enzyme analysis showed that the strain harbored enzymes that can completely degrade alginate and laminarin, which are the specific polysaccharides of brown algae. We also found genes for the utilization of mannitol, the major storage monosaccharide in the cell of brown algae. To understand the process of brown algae decomposition by B. weihaiensis Alg07, RNA-seq transcriptome analysis and qRT-PCR were performed. The genes involved in alginate metabolism were all up-regulated in the initial stage of kelp degradation, suggesting that the strain Alg07 first degrades alginate to destruct the cell wall so that the laminarin and mannitol are released and subsequently decomposed. The key genes involved in alginate and laminarin degradation were expressed in Escherichia coli and characterized. Overall, the model of brown algae degradation by the marine strain Alg07 was established, and novel alginate lyases and laminarinase were discovered.


July 7, 2019

Crystal structures of the TRIC trimeric intracellular cation channel orthologues.

Ca(2+) release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca(2+) signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.


July 7, 2019

Chimeras link to tandem repeats and transposable elements in tetraploid hybrid fish

Abstract The formation of the allotetraploid hybrid lineage (4nAT) encompasses both distant hybridization and polyploidization processes. The allotetraploid offspring have two sets of sub-genomes inherited from both parental species and therefore it is important to explore its genetic structure. Herein, we construct a bacterial artificial chromosome library of allotetraploids, and then sequence and analyze the full-length sequences of 19 bacterial artificial chromosomes. Sixty-eight DNA chimeras are identified, which are divided into four models according to the distribution of the genomic DNA derived from the parents. Among the 68 genetic chimeras, 44 (64.71%) are linked to tandem repeats (TRs) and 23 (33.82%) are linked to transposable elements (TEs). The chimeras linked to TRs are related to slipped-strand mispairing and double-strand break repair while the chimeras linked to TEs are benefit from the intervention of recombinases. In addition, TRs and TEs are linked not only with the recombinations, but also with the insertions/deletions of DNA segments. We conclude that DNA chimeras accompanied by TRs and TEs coordinate a balance between the sub-genomes derived from the parents which reduces the genomic shock effects and favors the evolutionary and adaptive capacity of the allotetraploidization. It is the first report on the relationship between formation of the DNA chimeras and TRs and TEs in the polyploid animals.


July 7, 2019

Whole genome analysis of Yersinia ruckeri isolated over 27 years in Australia and New Zealand reveals geographical endemism over multiple lineages and recent evolution under host selection.

Yersinia ruckeri is a salmonid pathogen with widespread distribution in cool-temperate waters including Australia and New Zealand, two isolated environments with recently developed salmonid farming industries. Phylogenetic comparison of 58 isolates from Australia, New Zealand, USA, Chile, Finland and China based on non-recombinant core genome SNPs revealed multiple deep-branching lineages, with a most recent common ancestor estimated at 18?500 years BP (12?355-24?757 95% HPD) and evidence of Australasian endemism. Evolution within the Tasmanian Atlantic salmon serotype O1b lineage has been slow, with 63 SNPs describing the variance over 27 years. Isolates from the prevailing lineage are poorly/non-motile compared to a lineage pre-vaccination, introduced in 1997, which is highly motile but has not been isolated since from epizootics. A non-motile phenotype has arisen independently in Tasmania compared to Europe and USA through a frameshift in fliI, encoding the ATPase of the flagella cluster. We report for the first time lipopolysaccharide O-antigen serotype O2 isolates in Tasmania. This phenotype results from deletion of the O-antigen cluster and consequent loss of high-molecular-weight O-antigen. This phenomenon has occurred independently on three occasions on three continents (Australasia, North America and Asia) as O2 isolates from the USA, China and Tasmania share the O-antigen deletion but occupy distant lineages. Despite the European and North American origins of the Australasian salmonid stocks, the lineages of Y. ruckeri in Australia and New Zealand are distinct from those of the northern hemisphere, suggesting they are pre-existing ancient strains that have emerged and evolved with the introduction of susceptible hosts following European colonization.


July 7, 2019

A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem.

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover, our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver.


July 7, 2019

Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast Saccharomycopsis fibuligera and its interspecies hybrid.

Genomic studies on fungal species with hydrolytic activity have gained increased attention due to their great biotechnological potential for biomass-based biofuel production. The amylolytic yeast Saccharomycopsis fibuligera has served as a good source of enzymes and genes involved in saccharification. Despite its long history of use in food fermentation and bioethanol production, very little is known about the basic physiology and genomic features of S. fibuligera.We performed whole-genome (WG) de novo sequencing and complete assembly of S. fibuligera KJJ81 and KPH12, two isolates from wheat-based Nuruk in Korea. Intriguingly, the KJJ81 genome (~38 Mb) was revealed as a hybrid between the KPH12 genome (~18 Mb) and another unidentified genome sharing 88.1% nucleotide identity with the KPH12 genome. The seven chromosome pairs of KJJ81 subgenomes exhibit highly conserved synteny, indicating a very recent hybridization event. The phylogeny inferred from WG comparisons showed an early divergence of S. fibuligera before the separation of the CTG and Saccharomycetaceae clades in the subphylum Saccharomycotina. Reconstructed carbon and sulfur metabolic pathways, coupled with RNA-Seq analysis, suggested a marginal Crabtree effect under high glucose and activation of sulfur metabolism toward methionine biosynthesis under sulfur limitation in this yeast. Notably, the lack of sulfate assimilation genes in the S. fibuligera genome reflects a unique phenotype for Saccharomycopsis clades as natural sulfur auxotrophs. Extended gene families, including novel genes involved in saccharification and proteolysis, were identified. Moreover, comparative genome analysis of S. fibuligera ATCC 36309, an isolate from chalky rye bread in Germany, revealed that an interchromosomal translocation occurred in the KPH12 genome before the generation of the KJJ81 hybrid genome.The completely sequenced S. fibuligera genome with high-quality annotation and RNA-Seq analysis establishes an important foundation for functional inference of S. fibuligera in the degradation of fermentation mash. The gene inventory facilitates the discovery of new genes applicable to the production of novel valuable enzymes and chemicals. Moreover, as the first gapless genome assembly in the genus Saccharomycopsis including members with desirable traits for bioconversion, the unique genomic features of S. fibuligera and its hybrid will provide in-depth insights into fungal genome dynamics as evolutionary adaptation.


July 7, 2019

Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen.

Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under ‘priming’ conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30?kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences – but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event – with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild.© 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.


July 7, 2019

Complete genome sequence of Aggregatibacter actinomycetemcomitans strain IDH781.

We report here the complete genomic sequence and methylome of Aggregatibacter actinomycetemcomitans strain IDH781. This rough strain is used extensively as a model organism to characterize localized aggressive periodontitis pathogenesis, the basic biology and oral cavity colonization of A. actinomycetemcomitans, and its interactions with other members of the oral microbiome. Copyright © 2016 May et al.


July 7, 2019

Cell cycle constraints and environmental control of local DNA hypomethylation in a-proteobacteria.

Heritable DNA methylation imprints are ubiquitous and underlie genetic variability from bacteria to humans. In microbial genomes, DNA methylation has been implicated in gene transcription, DNA replication and repair, nucleoid segregation, transposition and virulence of pathogenic strains. Despite the importance of local (hypo)methylation at specific loci, how and when these patterns are established during the cell cycle remains poorly characterized. Taking advantage of the small genomes and the synchronizability of a-proteobacteria, we discovered that conserved determinants of the cell cycle transcriptional circuitry establish specific hypomethylation patterns in the cell cycle model system Caulobacter crescentus. We used genome-wide methyl-N6-adenine (m6A-) analyses by restriction-enzyme-cleavage sequencing (REC-Seq) and single-molecule real-time (SMRT) sequencing to show that MucR, a transcriptional regulator that represses virulence and cell cycle genes in S-phase but no longer in G1-phase, occludes 5′-GANTC-3′ sequence motifs that are methylated by the DNA adenine methyltransferase CcrM. Constitutive expression of CcrM or heterologous methylases in at least two different a-proteobacteria homogenizes m6A patterns even when MucR is present and affects promoter activity. Environmental stress (phosphate limitation) can override and reconfigure local hypomethylation patterns imposed by the cell cycle circuitry that dictate when and where local hypomethylation is instated.


July 7, 2019

MICADo – Looking for mutations in targeted PacBio cancer data: an alignment-free method.

Targeted sequencing is commonly used in clinical application of NGS technology since it enables generation of sufficient sequencing depth in the targeted genes of interest and thus ensures the best possible downstream analysis. This notwithstanding, the accurate discovery and annotation of disease causing mutations remains a challenging problem even in such favorable context. The difficulty is particularly salient in the case of third generation sequencing technology, such as PacBio. We present MICADo, a de Bruijn graph based method, implemented in python, that makes possible to distinguish between patient specific mutations and other alterations for targeted sequencing of a cohort of patients. MICADo analyses NGS reads for each sample within the context of the data of the whole cohort in order to capture the differences between specificities of the sample with respect to the cohort. MICADo is particularly suitable for sequencing data from highly heterogeneous samples, especially when it involves high rates of non-uniform sequencing errors. It was validated on PacBio sequencing datasets from several cohorts of patients. The comparison with two widely used available tools, namely VarScan and GATK, shows that MICADo is more accurate, especially when true mutations have frequencies close to backgound noise. The source code is available at http://github.com/cbib/MICADo.


July 7, 2019

Complete genome anatomy of the emerging potato pathogen Dickeya solani type strain IPO 2222(T).

Several species of the genus Dickeya provoke soft rot and blackleg diseases on a wide range of plants and crops. Dickeya solani has been identified as the causative agent of diseases outbreaks on potato culture in Europe for the last decade. Here, we report the complete genome of the D. solani IPO 2222(T). Using PacBio and Illumina technologies, a unique circular chromosome of 4,919,833 bp was assembled. The G?+?C content reaches 56% and the genomic sequence contains 4,059 predicted proteins. The ANI values calculated for D. solani IPO 2222(T) vs. other available D. solani genomes was over 99.9% indicating a high genetic homogeneity within D. solani species.


July 7, 2019

Comparative genomics of Beauveria bassiana: uncovering signatures of virulence against mosquitoes.

Entomopathogenic fungi such as Beauveria bassiana are promising biological agents for control of malaria mosquitoes. Indeed, infection with B. bassiana reduces the lifespan of mosquitoes in the laboratory and in the field. Natural isolates of B. bassiana show up to 10-fold differences in virulence between the most and the least virulent isolate. In this study, we sequenced the genomes of five isolates representing the extremes of low/high virulence and three RNA libraries, and applied a genome comparison approach to uncover genetic mechanisms underpinning virulence.A high-quality, near-complete genome assembly was achieved for the highly virulent isolate Bb8028, which was compared to the assemblies of the four other isolates. Whole genome analysis showed a high level of genetic diversity between the five isolates (2.85-16.8 SNPs/kb), which grouped into two distinct phylogenetic clusters. Mating type gene analysis revealed the presence of either the MAT1-1-1 or the MAT1-2-1 gene. Moreover, a putative new MAT gene (MAT1-2-8) was detected in the MAT1-2 locus. Comparative genome analysis revealed that Bb8028 contains 163 genes exclusive for this isolate. These unique genes have a tendency to cluster in the genome and to be often located near the telomeres. Among the genes unique to Bb8028 are a Non-Ribosomal Peptide Synthetase (NRPS) secondary metabolite gene cluster, a polyketide synthase (PKS) gene, and five genes with homology to bacterial toxins. A survey of candidate virulence genes for B. bassiana is presented.Our results indicate several genes and molecular processes that may underpin virulence towards mosquitoes. Thus, the genome sequences of five isolates of B. bassiana provide a better understanding of the natural variation in virulence and will offer a major resource for future research on this important biological control agent.


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