Menu
April 21, 2020

Graph analysis of fragmented long-read bacterial genome assemblies.

Long-read genome assembly tools are expected to reconstruct bacterial genomes nearly perfectly, however they still produce fragmented assemblies in some cases. It would be beneficial to understand whether these cases are intrinsically impossible to resolve, or if assemblers are at fault, implying that genomes could be refined or even finished with little to no additional experimental cost.We propose a set of computational techniques to assist inspection of fragmented bacterial genome assemblies, through careful analysis of assembly graphs. By finding paths of overlapping raw reads between pairs of contigs, we recover potential short-range connections between contigs that were lost during the assembly process. We show that our procedure recovers 45% of missing contig adjacencies in fragmented Canu assemblies, on samples from the NCTC bacterial sequencing project. We also observe that a simple procedure based on enumerating weighted Hamiltonian cycles can suggest likely contig orderings. In our tests, the correct contig order is ranked first in half of the cases and within the top-3 predictions in nearly all evaluated cases, providing a direction for finishing fragmented long-read assemblies.https://gitlab.inria.fr/pmarijon/knot.Supplementary data are available at Bioinformatics online. © The Author(s) (2019). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.


April 21, 2020

Microbial diversity in the tick Argas japonicus (Acari: Argasidae) with a focus on Rickettsia pathogens.

The soft tick Argas japonicus mainly infests birds and can cause human dermatitis; however, no pathogen has been identified from this tick species in China. In the present study, the microbiota in A. japonicus collected from an epidemic community was explored, and some putative Rickettsia pathogens were further characterized. The results obtained indicated that bacteria in A. japonicus were mainly ascribed to the phyla Proteobacteria, Firmicutes and Actinobacteria. At the genus level, the male A. japonicus harboured more diverse bacteria than the females and nymphs. The bacteria Alcaligenes, Pseudomonas, Rickettsia and Staphylococcus were common in nymphs and adults. The abundance of bacteria belonging to the Rickettsia genus in females and males was 7.27% and 10.42%, respectively. Furthermore, the 16S rRNA gene of Rickettsia was amplified and sequenced, and phylogenetic analysis revealed that 13 sequences were clustered with the spotted fever group rickettsiae (Rickettsia heilongjiangensis and Rickettsia japonica) and three were clustered with Rickettsia limoniae, which suggested that the characterized Rickettsia in A. japonicus were novel putative pathogens and also that the residents were at considerable risk for infection by tick-borne pathogens. © 2019 The Royal Entomological Society.


April 21, 2020

Genome sequence resources for four phytopathogenic fungi from the Colletotrichum orbiculare species complex.

Colletotrichum orbiculare species complex fungi are hemibiotrophic plant pathogens that cause anthracnose of field crops and weeds. Members of this group have genomes that are remarkably expanded relative to other Colletotrichum fungi and compartmentalized into AT-rich, gene poor and GC-rich, gene rich regions. Here we present an updated version of the Colletotrichum orbiculare genome, as well as draft genomes of three other members from the C. orbiculare species complex; the alfalfa pathogen Colletotrichum trifolii, the prickly mallow pathogen Colletotrichum sidae and the burweed pathogen Colletotrichum spinosum. The data reported here will be important for comparative genomics analyses to identify factors that play a role in the evolution and maintenance of the expanded, compartmentalized genomes of these fungi which may contribute to their pathogenicity.


April 21, 2020

Pandemic spread of blaKPC-2 among Klebsiella pneumoniae ST11 in China is associated with horizontal transfer mediated by IncFII-like plasmids.

This study aimed to investigate the spread of the blaKPC-2 gene among Klebsiella pneumoniae and to illustrate the mechanism of dissemination of KPC-producing K. pneumoniae (KPC-Kp) ST11 in China.A total of 354 K. pneumoniae isolates were collected from four hospitals in China and were characterized by Multilocus sequence typing (MLST). Mobile genetic elements (MGEs) and pulsed-field gel electrophoresis (PFGE) analysis were used to identify the subtypes of K. pneumoniae ST11. PCR-based amplification and sequencing were performed to analyze Tn1721 transposons and IncFII-like plasmids. Electroporation experiments and whole-genome sequencing (WGS) analysis were used to reveal the genetic environment of the blaKPC-2 gene.As the primary type(87.1%) of KPC-Kp, K. pneumoniae ST11 was not predominant in nonKPC-Kp(3.1%). ST11 KPC-Kp was clonally heterogeneous and could be further classified into eleven MGE types and fourteen PFGE subtypes. Five Tn1721-blaKPC-2 variants were identified on IncFII-like plasmids. The detection rate of IncFII-like plasmids was much higher in ST11 KPC-Kp (100%) compared with non-ST11 KPC-Kp (16.0%) and the nonKPC-Kp group (7.5%). Moreover, the IncFII plasmid (with IIa replicon) was primarily detected on the MGE-F type (61.7%). The IncFIIk plasmid (with IIk replicon) was clustered into two subtypes: MGE-A (28.3%) and -F (41.5%). The detection of the IncFII and IncFIIk plasmids on MGE-A was 57.1% (20/35) and 42.9% (15/35), respectively.We revealed a close correlation between ST11 KPC-Kp and IncFII-like plasmids. Horizontal transfer mediated by IncFII-like plasmids plays an important role in the pandemic expansion of blaKPC-2 among K. pneumoniae ST11 in China. Copyright © 2019. Published by Elsevier B.V.


April 21, 2020

Enrichment of oral microbiota in early cystic precursors to invasive pancreatic cancer.

Intraductal papillary mucinous neoplasms (IPMNs) are pancreatic cysts that can progress to invasive pancreatic cancer. Associations between oncogenesis and oral microbiome alterations have been reported. This study aims to investigate a potential intracystic pancreatic microbiome in a pancreatic cystic neoplasm (PCN) surgery patient cohort.Paired cyst fluid and plasma were collected at pancreatic surgery from patients with suspected PCN (n=105). Quantitative and qualitative assessment of bacterial DNA by qPCR, PacBio sequencing (n=35), and interleukin (IL)-1ß quantification was performed. The data were correlated to diagnosis, lesion severity and clinical and laboratory profile, including proton-pump inhibitor (PPI) usage and history of invasive endoscopy procedures.Intracystic bacterial 16S DNA copy number and IL-1ß protein quantity were significantly higher in IPMN with high-grade dysplasia and IPMN with cancer compared with non-IPMN PCNs. Despite high interpersonal variation of intracystic microbiota composition, bacterial network and linear discriminant analysis effect size analyses demonstrated co-occurrence and enrichment of oral bacterial taxa including Fusobacterium nucleatum and Granulicatella adiacens in cyst fluid from IPMN with high-grade dysplasia. The elevated intracystic bacterial DNA is associated with, but not limited to, prior exposure to invasive endoscopic procedures, and is independent from use of PPI and antibiotics.Collectively, these findings warrant further investigation into the role of oral bacteria in cystic precursors to pancreatic cancer and have added values on the aetiopathology as well as the management of pancreatic cysts. © Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.


April 21, 2020

Mce3R Stress-Resistance Pathway Is Vulnerable to Small-Molecule Targeting That Improves Tuberculosis Drug Activities.

One-third of the world’s population carries Mycobacterium tuberculosis ( Mtb), the infectious agent that causes tuberculosis (TB), and every 17 s someone dies of TB. After infection, Mtb can live dormant for decades in a granuloma structure arising from the host immune response, and cholesterol is important for this persistence of Mtb. Current treatments require long-duration drug regimens with many associated toxicities, which are compounded by the high doses required. We phenotypically screened 35 6-azasteroid analogues against Mtb and found that, at low micromolar concentrations, a subset of the analogues sensitized Mtb to multiple TB drugs. Two analogues were selected for further study to characterize the bactericidal activity of bedaquiline and isoniazid under normoxic and low-oxygen conditions. These two 6-azasteroids showed strong synergy with bedaquiline (fractional inhibitory concentration index = 0.21, bedaquiline minimal inhibitory concentration = 16 nM at 1 µM 6-azasteroid). The rate at which spontaneous resistance to one of the 6-azasteroids arose in the presence of bedaquiline was approximately 10-9, and the 6-azasteroid-resistant mutants retained their isoniazid and bedaquiline sensitivity. Genes in the cholesterol-regulated Mce3R regulon were required for 6-azasteroid activity, whereas genes in the cholesterol catabolism pathway were not. Expression of a subset of Mce3R genes was down-regulated upon 6-azasteroid treatment. The Mce3R regulon is implicated in stress resistance and is absent in saprophytic mycobacteria. This regulon encodes a cholesterol-regulated stress-resistance pathway that we conclude is important for pathogenesis and contributes to drug tolerance, and this pathway is vulnerable to small-molecule targeting in live mycobacteria.


April 21, 2020

Defining transgene insertion sites and off-target effects of homology-based gene silencing informs the use of functional genomics tools in Phytophthora infestans.

DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spreads to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5′ ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for twelve different gene targets indicated that neighbors within 500-nt were often co-silenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, cis-spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not co-silenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in Phytophthora but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.


April 21, 2020

Biogeography and Microscale Diversity Shape the Biosynthetic Potential of Fungus-growing Ant-associated Pseudonocardia

The geographic and phylogenetic scale of ecologically relevant microbial diversity is still poorly understood. Using a model mutualism, fungus-growing ants and their defensive bacterial associate Pseudonocardia, we analyzed genetic diversity and biosynthetic potential in 46 strains isolated from ant colonies in a 20km transect near Barro Colorado Island in Panama. Despite an average pairwise core genome similarity of greater than 99%, population genomic analysis revealed several distinct bacterial populations matching ant host geographic distribution. We identified both genetic diversity signatures and divergent genes distinct to each lineage. We also identify natural product biosynthesis clusters specific to isolation locations. These geographic patterns were observable despite the populations living in close proximity to each other and provides evidence of ongoing genetic exchange. Our results add to the growing body of literature suggesting that variation in traits of interest can be found at extremely fine phylogenetic scales.


April 21, 2020

Variation in genome content and predatory phenotypes between Bdellovibrio sp. NC01 isolated from soil and B. bacteriovorus type strain HD100

The range of naturally occurring variation in the ability of Bdellovibrio strains to attack and kill Gram-negative bacteria is not well understood. Defining phenotypic and associated genotypic variation among Bdellovibrio may further our understanding of how this genus impacts microbial communities. In addition, comparisons of the predatory phenotypes of divergent strains may inform the development of Bdellovibrio as biocontrol agents to combat bacterial infections. We isolated Bdellovibrio sp. NC01 from soil and compared its genome and predatory phenotypes to B. bacteriovorus type strain HD100. Based on analysis of 16S rRNA gene sequences and average amino acid identity, NC01 belongs to a different species than HD100. Genome-wide comparisons and individual gene analyses indicated that eight NC01 genome regions were likely acquired by horizontal gene transfer (HGT), further supporting an important role for HGT in Bdellovibrio genome evolution. Within these regions, multiple protein-coding sequences were assigned predicted functions related to transcriptional regulation and transport; however, most were annotated as hypothetical proteins. Compared to HD100, NC01 has a limited prey range and kills E. coli ML35 less efficiently. Whereas HD100 drastically reduces the ML35 population and then maintains low prey population density, NC01 causes a smaller reduction in ML35, after which the prey population recovers, accompanied by a decrease in NC01. In addition, NC01 forms turbid plaques on lawns of E. coli ML35, in contrast to clear plaques formed by HD100. Characterizing variation in interactions between Bdellovibrio and Gram-negative bacteria, such as observed with NC01 and HD100, is valuable for understanding the ecological significance of predatory bacteria and evaluating their effectiveness in clinical applications.


April 21, 2020

Divergent selection following speciation in two ectoparasitic honey bee mites

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species Varroa destructor and Varroa jacobsoni. These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities.


April 21, 2020

SyRI: identification of syntenic and rearranged regions from whole-genome assemblies

We present SyRI, an efficient tool for genome-wide identification of structural rearrangements (SR) from genome graphs, which are built up from pair-wise whole-genome alignments. Instead of searching for differences, SyRI starts by finding all co-linear regions between the genomes. As all remaining regions are SRs by definition, they can be classified as inversions, translocations, or duplications based on their positions in convoluted networks of repetitive alignments. Finally, SyRI reports local variations like SNPs and indels within syntenic and rearranged regions. We show SyRItextquoterights broad applicability to multiple species and genetically validate the presence of ~100 translocations identified in Arabidopsis.


April 21, 2020

Draft Genome Assembly and Annotation of Red Raspberry Rubus Idaeus

The red raspberry, Rubus idaeus, is widely distributed in all temperate regions of Europe, Asia, and North America and is a major commercial fruit valued for its taste, high antioxidant and vitamin content. However, Rubus breeding is a long and slow process hampered by limited genomic and molecular resources. Genomic resources such as a complete genome sequencing and transcriptome will be of exceptional value to improve research and breeding of this high value crop. Using a hybrid sequence assembly approach including data from both long and short sequence reads, we present the first assembly of the Rubus idaeus genome (Joan J. variety). The de novo assembled genome consists of 2,145 scaffolds with a genome completeness of 95.3% and an N50 score of 638 KB. Leveraging a linkage map, we anchored 80.1% of the genome onto seven chromosomes. Using over 1 billion paired-end RNAseq reads, we annotated 35,566 protein coding genes with a transcriptome completeness score of 97.2%. The Rubus idaeus genome provides an important new resource for researchers and breeders.


April 21, 2020

The Ptr1 locus of Solanum lycopersicoides confers resistance to race 1 strains of Pseudomonas syringae pv. tomato and to Ralstonia pseudosolanacearum by recognizing the type III effectors AvrRpt2/RipBN.

Race 1 strains of Pseudomonas syringae pv. tomato, which cause bacterial speck disease of tomato, are becoming increasingly common and no simply-inherited genetic resistance to such strains is known. We discovered that a locus in Solanum lycopersicoides, termed Pseudomonas tomato race 1 (Ptr1), confers resistance to race 1 Pst strains by detecting the activity of type III effector AvrRpt2. In Arabidopsis, AvrRpt2 degrades the RIN4 protein thereby activating RPS2-mediated immunity. Using site-directed mutagenesis of AvrRpt2 we found that, like RPS2, activation of Ptr1 requires AvrRpt2 proteolytic activity. Ptr1 also detected the activity of AvrRpt2 homologs from diverse bacteria including one in Ralstonia pseudosolanacearum. The genome sequence of S. lycopersicoides revealed no RPS2 homolog in the Ptr1 region. Ptr1 could play an important role in controlling bacterial speck disease and its future cloning may shed light on an example of convergent evolution for recognition of a widespread type III effector.


April 21, 2020

Virus-host coexistence in phytoplankton through the genomic lens

Phytoplankton-virus interactions are major determinants of geochemical cycles in the oceans. Viruses are responsible for the redirection of carbon and nutrients away from larger organisms back towards microorganisms via the lysis of microalgae in a process coined the “viral shunt”. Virus-host interactions are generally expected to follow “boom and bust” dynamics, whereby a numerically dominant strain is lysed and replaced by a virus resistant strain. Here, we isolated a microalga and its infective nucleo-cytoplasmic large DNA virus (NCLDV) concomitantly from the environment in the surface NW Mediterranean Sea, Ostreococcus mediterraneus, and show continuous growth in culture of both the microalga and the virus. Evolution experiments through single cell bottlenecks demonstrate that, in the absence of the virus, susceptible cells evolve from one ancestral resistant single cell, and vice-versa; that is that resistant cells evolve from one ancestral susceptible cell. This provides evidence that the observed sustained viral production is the consequence of a minority of virus-susceptible cells. The emergence of these cells is explained by low-level phase switching between virus-resistant and virus-susceptible phenotypes, akin to a bet hedging strategy. Whole genome sequencing and analysis of the ~14 Mb microalga and the ~200 kb virus points towards ancient speciation of the microalga within the Ostreococcus species complex and frequent gene exchanges between prasinoviruses infecting Ostreococcus species. Re-sequencing of one susceptible strain demonstrated that the phase switch involved a large 60 Kb deletion of one chromosome. This chromosome is an outlier chromosome compared to the streamlined, gene dense, GC-rich standard chromosomes, as it contains many repeats and few orthologous genes. While this chromosome has been described in three different genera, its size increments have been previously associated to antiviral immunity and resistance in another species from the same genus. Mathematical modelling of this mechanism predicts microalga-virus population dynamics consistent with the observation of continuous growth of both virus and microalga. Altogether, our results suggest a previously overlooked strategy in phytoplankton-virus interactions.


April 21, 2020

Complete genome sequence of Bacillus velezensis JT3-1, a microbial germicide isolated from yak feces

Bacillus velezensis JT3-1 is a probiotic strain isolated from feces of the domestic yak (Bos grunniens) in the Gansu province of China. It has strong antagonistic activity against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella Typhimurium, Mannheimia haemolytica, Staphylococcus hominis, Clostridium perfringens, and Mycoplasma bovis. These properties have made the JT3-1 strain the focus of commercial interest. In this study, we describe the complete genome sequence of JT3-1, with a genome size of 3,929,799 bp, 3761 encoded genes and an average GC content of 46.50%. Whole genome sequencing of Bacillus velezensis JT3-1 will lay a good foundation for elucidation of the mechanisms of its antimicrobial activity, and for its future application.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.