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July 7, 2019

Identification of a Pseudomonas aeruginosa PAO1 DNA methyltransferase, its targets, and physiological roles.

DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We identified a conserved sequence motif targeted by an adenine methyltransferase of a type I R-M system and quantified the presence of N(6)-methyladenine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in the PAO1 methylation status were dependent on growth conditions and affected P. aeruginosa pathogenicity in a Galleria mellonella infection model. Furthermore, we found that methylated motifs in promoter regions led to shifts in sense and antisense gene expression, emphasizing the role of enzymatic DNA methylation as an epigenetic control of phenotypic traits in P. aeruginosa Since the DNA methylation enzymes are not encoded in the core genome, our findings illustrate how the acquisition of accessory genes can shape the global P. aeruginosa transcriptome and thus may facilitate adaptation to new and challenging habitats.IMPORTANCE With the introduction of advanced technologies, epigenetic regulation by DNA methyltransferases in bacteria has become a subject of intense studies. Here we identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif. The methylation level of all target sequences throughout the PAO1 genome was approximated to be in the range of 65 to 85% and was dependent on growth conditions. Inactivation of the methyltransferase revealed an attenuated-virulence phenotype in the Galleria mellonella infection model. Furthermore, differential expression of more than 90 genes was detected, including the small regulatory RNA prrF1, which contributes to a global iron-sparing response via the repression of a set of gene targets. Our finding of a methylation-dependent repression of the antisense transcript of the prrF1 small regulatory RNA significantly expands our understanding of the regulatory mechanisms underlying active DNA methylation in bacteria. Copyright © 2017 Doberenz et al.

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July 7, 2019

A pipeline for local assembly of minisatellite alleles from single-molecule sequencing data.

The advent of Next Generation Sequencing (NGS) has led to the generation of enormous volumes of short read sequence data, cheaply and in reasonable time scales. Nevertheless, the quality of genome assemblies generated using NGS technologies has been greatly affected, compared to those generated using Sanger DNA sequencing. This is largely due to the inability of short read sequence data to scaffold repetitive structures, creating gaps, inversions and rearrangements and resulting in assemblies that are, at best, draft forms. Third generation single-molecule sequencing (SMS) technologies (e.g. Pacific Biosciences Single Molecule Real Time (SMRT) system) address this challenge by generating sequences with increased read lengths, offering the prospect to better recover these complex repetitive structures, concomitantly improving assembly quality.Here, we evaluate the ability of SMS data (specifically human genome Pacific Biosciences SMRT data) to recover poorly represented repetitive sequences (specifically, GC-rich human minisatellites). To do this we designed a pipeline for the collection, processing and local assembly of single-molecule sequence data to form accurate contiguous local reconstructions. Our results show the recovery of an allele of the non-coding minisatellite MS1 (located on chromosome 1 at 1p33-35) at greater than 97% identity to reference (GRCh38) from the unprocessed sequence data of a haploid complete hydatidiform mole (CHM1) cell line. Furthermore, our assembly revealed an allele of over 500 repeat units; much larger than the reference (GRCh38), but consistent in structure with naturally occurring alleles that are segregating in human populations. This local assembly’s reconstruction was validated with the release of the whole genome assemblies GCA_001297185.1 and GCA_000772585.3, where this allele occurs. Additionally, application of this pipeline to coding minisatellites in the PRDM9 and ZNF93 genes enabled recovery of high identity allele structures for these sequence regions whose length was confirmed by PCR from cell line genomic DNA. The internal repeat structure of the PRDM9 allele recovered was consistent with common human-specific alleles.Code available at https://github.com/ndliberial/smrt_pipeline CONTACT: dno2@le.ac.uk. © The Author 2016. Published by Oxford University Press.

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July 7, 2019

An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence.

Some of the bacterial cells in isogenic populations behave differently from others. We describe here how a new type of phenotypic heterogeneity relating to resistance to cationic antimicrobial peptides (CAMPs) is determinant for the pathogenic infection process of the entomopathogenic bacterium Photorhabdus luminescens. We demonstrate that the resistant subpopulation, which accounts for only 0.5% of the wild-type population, causes septicemia in insects. Bacterial heterogeneity is driven by the PhoPQ two-component regulatory system and expression of pbgPE, an operon encoding proteins involved in lipopolysaccharide (LPS) modifications. We also report the characterization of a core regulon controlled by the DNA-binding PhoP protein, which governs virulence in P. luminescens. Comparative RNAseq analysis revealed an upregulation of marker genes for resistance, virulence and bacterial antagonism in the pre-existing resistant subpopulation, suggesting a greater ability to infect insect prey and to survive in cadavers. Finally, we suggest that the infection process of P. luminescens is based on a bet-hedging strategy to cope with the diverse environmental conditions experienced during the lifecycle.

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July 7, 2019

Innovations and challenges in detecting long read overlaps: an evaluation of the state-of-the-art.

Identifying overlaps between error-prone long reads, specifically those from Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PB), is essential for certain downstream applications, including error correction and de novo assembly. Though akin to the read-to-reference alignment problem, read-to-read overlap detection is a distinct problem that can benefit from specialized algorithms that perform efficiently and robustly on high error rate long reads. Here, we review the current state-of-the-art read-to-read overlap tools for error-prone long reads, including BLASR, DALIGNER, MHAP, GraphMap and Minimap. These specialized bioinformatics tools differ not just in their algorithmic designs and methodology, but also in their robustness of performance on a variety of datasets, time and memory efficiency and scalability. We highlight the algorithmic features of these tools, as well as their potential issues and biases when utilizing any particular method. To supplement our review of the algorithms, we benchmarked these tools, tracking their resource needs and computational performance, and assessed the specificity and precision of each. In the versions of the tools tested, we observed that Minimap is the most computationally efficient, specific and sensitive method on the ONT datasets tested; whereas GraphMap and DALIGNER are the most specific and sensitive methods on the tested PB datasets. The concepts surveyed may apply to future sequencing technologies, as scalability is becoming more relevant with increased sequencing throughput.cjustin@bcgsc.ca , ibirol@bcgsc.ca.Supplementary data are available at Bioinformatics online.

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July 7, 2019

Genomic innovation for crop improvement.

Crop production needs to increase to secure future food supplies, while reducing its impact on ecosystems. Detailed characterization of plant genomes and genetic diversity is crucial for meeting these challenges. Advances in genome sequencing and assembly are being used to access the large and complex genomes of crops and their wild relatives. These have helped to identify a wide spectrum of genetic variation and permitted the association of genetic diversity with diverse agronomic phenotypes. In combination with improved and automated phenotyping assays and functional genomic studies, genomics is providing new foundations for crop-breeding systems.

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July 7, 2019

Fungal volatile compounds induce production of the secondary metabolite Sodorifen in Serratia plymuthica PRI-2C.

The ability of bacteria and fungi to communicate with each other is a remarkable aspect of the microbial world. It is recognized that volatile organic compounds (VOCs) act as communication signals, however the molecular responses by bacteria to fungal VOCs remain unknown. Here we perform transcriptomics and proteomics analyses of Serratia plymuthica PRI-2C exposed to VOCs emitted by the fungal pathogen Fusarium culmorum. We find that the bacterium responds to fungal VOCs with changes in gene and protein expression related to motility, signal transduction, energy metabolism, cell envelope biogenesis, and secondary metabolite production. Metabolomic analysis of the bacterium exposed to the fungal VOCs, gene cluster comparison, and heterologous co-expression of a terpene synthase and a methyltransferase revealed the production of the unusual terpene sodorifen in response to fungal VOCs. These results strongly suggest that VOCs are not only a metabolic waste but important compounds in the long-distance communication between fungi and bacteria.

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July 7, 2019

The Nephila clavipes genome highlights the diversity of spider silk genes and their complex expression.

Spider silks are the toughest known biological materials, yet are lightweight and virtually invisible to the human immune system, and they thus have revolutionary potential for medicine and industry. Spider silks are largely composed of spidroins, a unique family of structural proteins. To investigate spidroin genes systematically, we constructed the first genome of an orb-weaving spider: the golden orb-weaver (Nephila clavipes), which builds large webs using an extensive repertoire of silks with diverse physical properties. We cataloged 28 Nephila spidroins, representing all known orb-weaver spidroin types, and identified 394 repeated coding motif variants and higher-order repetitive cassette structures unique to specific spidroins. Characterization of spidroin expression in distinct silk gland types indicates that glands can express multiple spidroin types. We find evidence of an alternatively spliced spidroin, a spidroin expressed only in venom glands, evolutionary mechanisms for spidroin diversification, and non-spidroin genes with expression patterns that suggest roles in silk production.

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July 7, 2019

Sequencing and de novo assembly of a near complete indica rice genome.

A high-quality reference genome is critical for understanding genome structure, genetic variation and evolution of an organism. Here we report the de novo assembly of an indica rice genome Shuhui498 (R498) through the integration of single-molecule sequencing and mapping data, genetic map and fosmid sequence tags. The 390.3?Mb assembly is estimated to cover more than 99% of the R498 genome and is more continuous than the current reference genomes of japonica rice Nipponbare (MSU7) and Arabidopsis thaliana (TAIR10). We annotate high-quality protein-coding genes in R498 and identify genetic variations between R498 and Nipponbare and presence/absence variations by comparing them to 17 draft genomes in cultivated rice and its closest wild relatives. Our results demonstrate how to de novo assemble a highly contiguous and near-complete plant genome through an integrative strategy. The R498 genome will serve as a reference for the discovery of genes and structural variations in rice.

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July 7, 2019

Regulation of PI-2b pilus expression in hypervirulent Streptococcus agalactiae ST-17 BM110.

The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) “hypervirulent” ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3- to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfp as reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5′ promoter region is required for maximal transcription of gfp as a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110?covR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential.

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July 7, 2019

De novo genome and transcriptome assembly of the Canadian beaver (Castor canadensis).

The Canadian beaver (Castor canadensis) is the largest indigenous rodent in North America. We report a draft annotated assembly of the beaver genome, the first for a large rodent and the first mammalian genome assembled directly from uncorrected and moderate coverage (< 30 ×) long reads generated by single-molecule sequencing. The genome size is 2.7 Gb estimated by k-mer analysis. We assembled the beaver genome using the new Canu assembler optimized for noisy reads. The resulting assembly was refined using Pilon supported by short reads (80 ×) and checked for accuracy by congruency against an independent short read assembly. We scaffolded the assembly using the exon-gene models derived from 9805 full-length open reading frames (FL-ORFs) constructed from the beaver leukocyte and muscle transcriptomes. The final assembly comprised 22,515 contigs with an N50 of 278,680 bp and an N50-scaffold of 317,558 bp. Maximum contig and scaffold lengths were 3.3 and 4.2 Mb, respectively, with a combined scaffold length representing 92% of the estimated genome size. The completeness and accuracy of the scaffold assembly was demonstrated by the precise exon placement for 91.1% of the 9805 assembled FL-ORFs and 83.1% of the BUSCO (Benchmarking Universal Single-Copy Orthologs) gene set used to assess the quality of genome assemblies. Well-represented were genes involved in dentition and enamel deposition, defining characteristics of rodents with which the beaver is well-endowed. The study provides insights for genome assembly and an important genomics resource for Castoridae and rodent evolutionary biology. Copyright © 2017 Lok et al.

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July 7, 2019

Genome sequence of the fungal strain 14919 producing 3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitor FR901512.

Fungal strain 14919 was originally isolated from a soil sample collected at Mt. Kiyosumi, Chiba Prefecture, Japan. It produces FR901512, a potent and strong 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor. The genome sequence of fungal strain 14919 was determined and annotated to improve the productivity of FR901512. Copyright © 2017 Itoh et al.

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July 7, 2019

The evolutionary life cycle of the polysaccharide biosynthetic gene cluster based on the Sphingomonadaceae.

Although clustering of genes from the same metabolic pathway is a widespread phenomenon, the evolution of the polysaccharide biosynthetic gene cluster remains poorly understood. To determine the evolution of this pathway, we identified a scattered production pathway of the polysaccharide sanxan by Sphingomonas sanxanigenens NX02, and compared the distribution of genes between sphingan-producing and other Sphingomonadaceae strains. This allowed us to determine how the scattered sanxan pathway developed, and how the polysaccharide gene cluster evolved. Our findings suggested that the evolution of microbial polysaccharide biosynthesis gene clusters is a lengthy cyclic process comprising cluster 1???scatter???cluster 2. The sanxan biosynthetic pathway proved the existence of a dispersive process. We also report the complete genome sequence of NX02, in which we identified many unstable genetic elements and powerful secretion systems. Furthermore, nine enzymes for the formation of activated precursors, four glycosyltransferases, four acyltransferases, and four polymerization and export proteins were identified. These genes were scattered in the NX02 genome, and the positive regulator SpnA of sphingans synthesis could not regulate sanxan production. Finally, we concluded that the evolution of the sanxan pathway was independent. NX02 evolved naturally as a polysaccharide producing strain over a long-time evolution involving gene acquisitions and adaptive mutations.

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July 7, 2019

Genome sequencing and population genomic analyses provide insights into the adaptive landscape of silver birch.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

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July 7, 2019

High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development.

Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development.

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July 7, 2019

Transcriptome Remodeling of Acinetobacter baumannii during Infection and Treatment.

Acinetobacter baumannii is an increasingly common multidrug-resistant pathogen in health care settings. Although the genetic basis of antibiotic resistance mechanisms has been extensively studied, much less is known about how genetic variation contributes to other aspects of successful infections. Genetic changes that occur during host infection and treatment have the potential to remodel gene expression patterns related to resistance and pathogenesis. Longitudinal sets of multidrug-resistant A. baumannii isolates from eight patients were analyzed by RNA sequencing (RNA-seq) to identify differentially expressed genes and link them to genetic changes contributing to transcriptional variation at both within-patient and population levels. The number of differentially expressed genes among isolates from the same patient ranged from 26 (patient 588) to 145 (patient 475). Multiple patients had isolates with differential gene expression patterns related to mutations in the pmrAB and adeRS two-component regulatory system genes, as well as significant differences in genes related to antibiotic resistance, iron acquisition, amino acid metabolism, and surface-associated proteins. Population level analysis revealed 39 genetic regions with clade-specific differentially expressed genes, for which 19, 8, and 3 of these could be explained by insertion sequence mobilization, recombination-driven sequence variation, and intergenic mutations, respectively. Multiple types of mutations that arise during infection can significantly remodel the expression of genes that are known to be important in pathogenesis. IMPORTANCE Health care-associated multidrug-resistant Acinetobacter baumannii can cause persistent infections in patients, but bacterial cells must overcome host defenses and antibiotic therapies to do so. Genetic variation arises during host infection, and new mutations are often enriched in genes encoding transcriptional regulators, iron acquisition systems, and surface-associated structures. In this study, genetic variation was shown to result in transcriptome remodeling at the level of individual patients and across phylogenetic groups. Differentially expressed genes include those related to capsule modification, iron acquisition, type I pili, and antibiotic resistance. Population level transcriptional variation reflects genome dynamics over longer evolutionary time periods, and convergent transcriptional changes support the adaptive significance of these regions. Transcriptional changes can be attributed to multiple types of genomic change, but insertion sequence mobilization had a predominant effect. The transcriptional effects of mutations that arise during infection highlight the rapid adaptation of A. baumannii during host exposure. Copyright © 2017 Wright et al.

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