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July 7, 2019

Bacteriophages are the major drivers of Shigella flexneri serotype 1c genome plasticity: a complete genome analysis.

Shigella flexneri is the primary cause of bacillary dysentery in the developing countries. S. flexneri serotype 1c is a novel serotype, which is found to be endemic in many developing countries, but little is known about its genomic architecture and virulence signatures. We have sequenced for the first time, the complete genome of S. flexneri serotype 1c strain Y394, to provide insights into its diversity and evolution.We generated a high-quality reference genome of S. flexneri serotype 1c using the hybrid methods of long-read single-molecule real-time (SMRT) sequencing technology and short-read MiSeq (Illumina) sequencing technology. The Y394 chromosome is 4.58 Mb in size and shares the basic genomic features with other S. flexneri complete genomes. However, it possesses unique and highly modified O-antigen structure comprising of three distinct O-antigen modifying gene clusters that potentially came from three different bacteriophages. It also possesses a large number of hypothetical unique genes compared to other S. flexneri genomes.Despite a high level of structural and functional similarities of Y394 genome with other S. flexneri genomes, there are marked differences in the pathogenic islands. The diversity in the pathogenic islands suggests that these bacterial pathogens are well adapted to respond to the selection pressures during their evolution, which might contribute to the differences in their virulence potential.

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July 7, 2019

Root-associated fungal microbiota of nonmycorrhizal Arabis alpina and its contribution to plant phosphorus nutrition.

Most land plants live in association with arbuscular mycorrhizal (AM) fungi and rely on this symbiosis to scavenge phosphorus (P) from soil. The ability to establish this partnership has been lost in some plant lineages like the Brassicaceae, which raises the question of what alternative nutrition strategies such plants have to grow in P-impoverished soils. To understand the contribution of plant-microbiota interactions, we studied the root-associated fungal microbiome of Arabis alpina (Brassicaceae) with the hypothesis that some of its components can promote plant P acquisition. Using amplicon sequencing of the fungal internal transcribed spacer 2, we studied the root and rhizosphere fungal communities of A. alpina growing under natural and controlled conditions including low-P soils and identified a set of 15 fungal taxa consistently detected in its roots. This cohort included a Helotiales taxon exhibiting high abundance in roots of wild A. alpina growing in an extremely P-limited soil. Consequently, we isolated and subsequently reintroduced a specimen from this taxon into its native P-poor soil in which it improved plant growth and P uptake. The fungus exhibited mycorrhiza-like traits including colonization of the root endosphere and P transfer to the plant. Genome analysis revealed a link between its endophytic lifestyle and the expansion of its repertoire of carbohydrate-active enzymes. We report the discovery of a plant-fungus interaction facilitating the growth of a nonmycorrhizal plant under native P-limited conditions, thus uncovering a previously underestimated role of root fungal microbiota in P cycling.

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July 7, 2019

Parallel evolution of two clades of a major Atlantic endemic Vibrio parahaemolyticus pathogen lineage by independent acquisition of related pathogenicity islands.

Shellfish-transmitted Vibrio parahaemolyticus infections have recently increased from locations with historically low disease incidence, such as the Northeast United States (US). This change coincided with a bacterial population shift towards human pathogenic variants occurring in part through the introduction of several Pacific native lineages (ST36, ST43 and ST636) to near-shore areas off the Atlantic coast of the Northeast US. Concomitantly, ST631 emerged as a major endemic pathogen. Phylogenetic trees of clinical and environmental isolates indicated that two clades diverged from a common ST631 ancestor, and in each of these clades, a human pathogenic variant evolved independently through acquisition of distinct Vibrio pathogenicity islands (VPaI). These VPaI differ from each other and bear little resemblance to hemolysin-containing VPaI from isolates of the pandemic clonal complex. Clade I ST631 isolates either harbored no hemolysins, or contained a chromosome I-inserted island we call VPaIß that encodes a type three secretion system (T3SS2ß) typical of Trh hemolysin-producers. The more clinically prevalent and clonal ST631 clade II had an island we call VPaI? that encodes both tdh and trh and that was inserted in chromosome II. VPaI? was derived from VPaIß but with some additional acquired elements in common with VPaI carried by pandemic isolates, exemplifying the mosaic nature of pathogenicity islands. Genomics comparisons and amplicon assays identified VPaI?-type islands containing tdh inserted adjacent to the ure cluster in the three introduced Pacific and most other emergent lineages. that collectively cause 67% of Northeast US infections as of 2016.IMPORTANCE The availability of three different hemolysin genotypes in the ST631 lineage provided a unique opportunity to employ genome comparisons to further our understanding of the processes underlying pathogen evolution. The fact that two different pathogenic clades arose in parallel from the same potentially benign lineage by independent VPaI acquisition is surprising considering the historically low prevalence of community members harboring VPaI in waters along the Northeast US coast that could serve as the source of this material. This illustrates a possible predisposition of some lineages to not only acquire foreign DNA but also to become human pathogens. Whereas the underlying cause for the expansion of V. parahaemolyticus lineages harboring VPaI? along the US Atlantic coast and spread of this element to multiple lineages that underlies disease emergence is not known, this work underscores the need to define the environment factors that favor bacteria harboring VPaI in locations of emergent disease. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

SVachra: a tool to identify genomic structural variation in mate pair sequencing data containing inward and outward facing reads.

Characterization of genomic structural variation (SV) is essential to expanding the research and clinical applications of genome sequencing. Reliance upon short DNA fragment paired end sequencing has yielded a wealth of single nucleotide variants and internal sequencing read insertions-deletions, at the cost of limited SV detection. Multi-kilobase DNA fragment mate pair sequencing has supplemented the void in SV detection, but introduced new analytic challenges requiring SV detection tools specifically designed for mate pair sequencing data. Here, we introduce SVachra – Structural Variation Assessment of CHRomosomal Aberrations, a breakpoint calling program that identifies large insertions-deletions, inversions, inter- and intra-chromosomal translocations utilizing both inward and outward facing read types generated by mate pair sequencing.We demonstrate SVachra’s utility by executing the program on large-insert (Illumina Nextera) mate pair sequencing data from the personal genome of a single subject (HS1011). An additional data set of long-read (Pacific BioSciences RSII) was also generated to validate SV calls from SVachra and other comparison SV calling programs. SVachra exhibited the highest validation rate and reported the widest distribution of SV types and size ranges when compared to other SV callers.SVachra is a highly specific breakpoint calling program that exhibits a more unbiased SV detection methodology than other callers.

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July 7, 2019

Complete genome sequence of the fruiting myxobacterium Myxococcus macrosporus strain DSM 14697, generated by PacBio sequencing.

Members of the Myxococcales order initiate a developmental program in response to starvation that culminates in formation of spore-filled fruiting bodies. To investigate the genetic basis for fruiting body formation, we present the complete 8.9-Mb genome sequence of Myxococcus macrosporus strain DSM 14697, generated using the PacBio sequencing platform. Copyright © 2017 Treuner-Lange et al.

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July 7, 2019

The rapid in vivo evolution of Pseudomonas aeruginosa in ventilator-associated pneumonia patients leads to attenuated virulence.

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe airway infections in humans. These infections are usually difficult to treat and associated with high mortality rates. While colonizing the human airways, P. aeruginosa could accumulate genetic mutations that often lead to its better adaptability to the host environment. Understanding these evolutionary traits may provide important clues for the development of effective therapies to treat P. aeruginosa infections. In this study, 25 P. aeruginosa isolates were longitudinally sampled from the airways of four ventilator-associated pneumonia (VAP) patients. Pacbio and Illumina sequencing were used to analyse the in vivo evolutionary trajectories of these isolates. Our analysis showed that positive selection dominantly shaped P. aeruginosa genomes during VAP infections and led to three convergent evolution events, including loss-of-function mutations of lasR and mpl, and a pyoverdine-deficient phenotype. Specifically, lasR encodes one of the major transcriptional regulators in quorum sensing, whereas mpl encodes an enzyme responsible for recycling cell wall peptidoglycan. We also found that P. aeruginosa isolated at late stages of VAP infections produce less elastase and are less virulent in vivo than their earlier isolated counterparts, suggesting the short-term in vivo evolution of P. aeruginosa leads to attenuated virulence.© 2017 The Authors.

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July 7, 2019

Genetic plasticity of the Shigella virulence plasmid is mediated by intra- and inter-molecular events between insertion sequences.

Acquisition of a single copy, large virulence plasmid, pINV, led to the emergence of Shigella spp. from Escherichia coli. The plasmid encodes a Type III secretion system (T3SS) on a 30 kb pathogenicity island (PAI), and is maintained in a bacterial population through a series of toxin:antitoxin (TA) systems which mediate post-segregational killing (PSK). The T3SS imposes a significant cost on the bacterium, and strains which have lost the plasmid and/or genes encoding the T3SS grow faster than wild-type strains in the laboratory, and fail to bind the indicator dye Congo Red (CR). Our aim was to define the molecular events in Shigella flexneri that cause loss of Type III secretion (T3S), and to examine whether TA systems exert positional effects on pINV. During growth at 37°C, we found that deletions of regions of the plasmid including the PAI lead to the emergence of CR-negative colonies; deletions occur through intra-molecular recombination events between insertion sequences (ISs) flanking the PAI. Furthermore, by repositioning MvpAT (which belongs to the VapBC family of TA systems) near the PAI, we demonstrate that the location of this TA system alters the rearrangements that lead to loss of T3S, indicating that MvpAT acts both globally (by reducing loss of pINV through PSK) as well as locally (by preventing loss of adjacent sequences). During growth at environmental temperatures, we show for the first time that pINV spontaneously integrates into different sites in the chromosome, and this is mediated by inter-molecular events involving IS1294. Integration leads to reduced PAI gene expression and impaired secretion through the T3SS, while excision of pINV from the chromosome restores T3SS function. Therefore, pINV integration provides a reversible mechanism for Shigella to circumvent the metabolic burden imposed by pINV. Intra- and inter-molecular events between ISs, which are abundant in Shigella spp., mediate plasticity of S. flexneri pINV.

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July 7, 2019

Whole-genome sequences of bacteremia isolates of Bordetella holmesii.

Bordetella holmesii causes respiratory and invasive diseases in humans, but its pathogenesis remains poorly understood. We report here the genome sequences of seven bacteremia isolates of B. holmesii, including the type strain. Comparative analysis of these sequences may aid studies of B. holmesii biology and assist in the development of species-specific diagnostic strategies. Copyright © 2017 Tettelin et al.

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July 7, 2019

Evolution of sphingomonad gene clusters related to pesticide catabolism revealed by genome sequence and mobilomics of Sphingobium herbicidovorans MH.

Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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July 7, 2019

Genome sequence and composition of a tolyporphin-producing cyanobacterium-microbial community.

The cyanobacterial culture HT-58-2 was originally described as a strain of Tolypothrix nodosa with the ability to produce tolyporphins, which comprise a family of distinct tetrapyrrole macrocycles with reported efflux pump inhibition properties. Upon reviving the culture from what was thought to be a nonextant collection, studies of culture conditions, strain characterization, phylogeny, and genomics have been undertaken. Here, HT-58-2 was shown by 16S rRNA analysis to closely align with Brasilonema strains and not with Tolypothrix isolates. Light, fluorescence, and scanning electron microscopy revealed cyanobacterium filaments that are decorated with attached bacteria and associated with free bacteria. Metagenomic surveys of HT-58-2 cultures revealed a diversity of bacteria dominated by Erythrobacteraceae, 97% of which are Porphyrobacter species. A dimethyl sulfoxide washing procedure was found to yield enriched cyanobacterial DNA (presumably by removing community bacteria) and sequence data sufficient for genome assembly. The finished, closed HT-58-2Cyano genome consists of 7.85 Mbp (42.6% G+C) and contains 6,581 genes. All genes for biosynthesis of tetrapyrroles (e.g., heme, chlorophyll a, and phycocyanobilin) and almost all for cobalamin were identified dispersed throughout the chromosome. Among the 6,177 protein-encoding genes, coding sequences (CDSs) for all but two of the eight enzymes for conversion of glutamic acid to protoporphyrinogen IX also were found within one major gene cluster. The cluster also includes 10 putative genes (and one hypothetical gene) encoding proteins with domains for a glycosyltransferase, two cytochrome P450 enzymes, and a flavin adenine dinucleotide (FAD)-binding protein. The composition of the gene cluster suggests a possible role in tolyporphin biosynthesis. IMPORTANCE A worldwide search more than 25 years ago for cyanobacterial natural products with anticancer activity identified a culture (HT-58-2) from Micronesia that produces tolyporphins. Tolyporphins are tetrapyrroles, like chlorophylls, but have several profound structural differences that reside outside the bounds of known biosynthetic pathways. To begin probing the biosynthetic origin and biological function of tolyporphins, our research has focused on studying the cyanobacterial strain, about which almost nothing has been previously reported. We find that the HT-58-2 culture is composed of the cyanobacterium and a community of associated bacteria, complicating the question of which organisms make tolyporphins. Elucidation of the cyanobacterial genome revealed an intriguing gene cluster that contains tetrapyrrole biosynthesis genes and a collection of unknown genes, suggesting that the cluster may be responsible for tolyporphin production. Knowledge of the genome and the gene cluster sharply focuses research to identify related cyanobacterial producers of tolyporphins and delineate the tolyporphin biosynthetic pathway. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Systems biotechnology for protein production in Pichia pastoris.

The methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) is one of the most important production systems for heterologous proteins. After the first genome sequences were published in 2009, tremendous effort was made to establish systems-level analytical methods. Methylotrophic lifestyle was one of the most thoroughly investigated topics, studied at the levels of transcriptome, proteome and metabolic flux. Also the responses of P. pastoris to environmental stress conditions experienced during high cell density production processes were studied. Metabolomics and flux analysis revealed the plasticity of the cellular metabolism in its adaption to the production of foreign proteins and served as blueprints for subsequent cell engineering and/or process design. The transcriptional response elicited by overexpression of heterologous proteins seems to depend on the nature and complexity of the recombinant product. Based on these data, novel targets for strain engineering could be deduced from transcriptomics and proteomics data mining and effectively enhanced protein secretion. Transcriptional regulation data also served as a valuable resource to identify novel promoters with the desired regulatory characteristics. This review aims to provide a comprehensive overview of systems biology applications in P. pastoris ranging from increased understanding of cell physiology to improving recombinant protein production in this cell factory.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Natural product diversity associated with the nematode symbionts Photorhabdus and Xenorhabdus.

Xenorhabdus and Photorhabdus species dedicate a large amount of resources to the production of specialized metabolites derived from non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS). Both bacteria undergo symbiosis with nematodes, which is followed by an insect pathogenic phase. So far, the molecular basis of this tripartite relationship and the exact roles that individual metabolites and metabolic pathways play have not been well understood. To close this gap, we have significantly expanded the database for comparative genomics studies in these bacteria. Clustering the genes encoded in the individual genomes into hierarchical orthologous groups reveals a high-resolution picture of functional evolution in this clade. It identifies groups of genes-many of which are involved in secondary metabolite production-that may account for the niche specificity of these bacteria. Photorhabdus and Xenorhabdus appear very similar at the DNA sequence level, which indicates their close evolutionary relationship. Yet, high-resolution mass spectrometry analyses reveal a huge chemical diversity in the two taxa. Molecular network reconstruction identified a large number of previously unidentified metabolite classes, including the xefoampeptides and tilivalline. Here, we apply genomic and metabolomic methods in a complementary manner to identify and elucidate additional classes of natural products. We also highlight the ability to rapidly and simultaneously identify potentially interesting bioactive products from NRPSs and PKSs, thereby augmenting the contribution of molecular biology techniques to the acceleration of natural product discovery.

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July 7, 2019

A high-quality genome assembly of quinoa provides insights into the molecular basis of salt bladder-based salinity tolerance and the exceptional nutritional value.

Chenopodium quinoa is a halophytic pseudocereal crop that is being cultivated in an ever-growing number of countries. Because quinoa is highly resistant to multiple abiotic stresses and its seed has a better nutritional value than any other major cereals, it is regarded as a future crop to ensure global food security. We generated a high-quality genome draft using an inbred line of the quinoa cultivar Real. The quinoa genome experienced one recent genome duplication about 4.3 million years ago, likely reflecting the genome fusion of two Chenopodium parents, in addition to the ? paleohexaploidization reported for most eudicots. The genome is highly repetitive (64.5% repeat content) and contains 54 438 protein-coding genes and 192 microRNA genes, with more than 99.3% having orthologous genes from glycophylic species. Stress tolerance in quinoa is associated with the expansion of genes involved in ion and nutrient transport, ABA homeostasis and signaling, and enhanced basal-level ABA responses. Epidermal salt bladder cells exhibit similar characteristics as trichomes, with a significantly higher expression of genes related to energy import and ABA biosynthesis compared with the leaf lamina. The quinoa genome sequence provides insights into its exceptional nutritional value and the evolution of halophytes, enabling the identification of genes involved in salinity tolerance, and providing the basis for molecular breeding in quinoa.

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July 7, 2019

Building a locally diploid genome and transcriptome of the diatom Fragilariopsis cylindrus.

The genome of the cold-adapted diatom Fragilariopsis cylindrus is characterized by highly diverged haplotypes that intersperse its homozygous genome. Here, we describe how a combination of PacBio DNA and Illumina RNA sequencing can be used to resolve this complex genomic landscape locally into the highly diverged haplotypes, and how to map various environmentally controlled transcripts onto individual haplotypes. We assembled PacBio sequence data with the FALCON assembler and created a haplotype resolved annotation of the assembly using annotations of a Sanger sequenced F. cylindrus genome. RNA-seq datasets from six different growth conditions were used to resolve allele-specifc gene expression in F. cylindrus. This approach enables to study differential expression of alleles in a complex genomic landscape and provides a useful tool to study how diverged haplotypes in diploid organisms are used for adaptation and evolution to highly variable environments.

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July 7, 2019

Revealing the saline adaptation strategies of the halophilic bacterium Halomonas beimenensis through high-throughput omics and transposon mutagenesis approaches.

Studies on the halotolerance of bacteria are attractive to the fermentation industry. However, a lack of sufficient genomic information has precluded an investigation of the halotolerance of Halomonas beimenensis. Here, we describe the molecular mechanisms of saline adaptation in H. beimenensis based on high-throughput omics and Tn5 transposon mutagenesis. The H. beimenensis genome is 4.05 Mbp and contains 3,807 genes, which were sequenced using short and long reads obtained via deep sequencing. Sixteen Tn5 mutants with a loss of halotolerance were identified. Orthologs of the mutated genes, such as nqrA, trkA, atpC, nadA, and gdhB, have significant biological functions in sodium efflux, potassium uptake, hydrogen ion transport for energy conversion, and compatible solute synthesis, which are known to control halotolerance. Other genes, such as spoT, prkA, mtnN, rsbV, lon, smpB, rfbC, rfbP, tatB, acrR1, and lacA, function in cellular signaling, quorum sensing, transcription/translation, and cell motility also shown critical functions for promoting a halotolerance. In addition, KCl application increased halotolerance and potassium-dependent cell motility in a high-salinity environment. Our results demonstrated that a combination of omics and mutagenesis could be used to facilitate the mechanistic exploitation of saline adaptation in H. beimenensis, which can be applied for biotechnological purposes.

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