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Auto Tag: De novo assembly

September 22, 2019

Endogenous rRNA sequence variation can regulate stress response gene expression and phenotype.

Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, RelA and RelE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

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September 22, 2019

Comparative genome analysis and evaluation of probiotic characteristics of Lactobacillus plantarum strain JDFM LP11.

In the current study, the probiotic potential of approximately 250 strains of lactic acid bacteria (LAB) isolated from piglet fecal samples were investigated; among them Lactobacillus plantarum strain JDFM LP11, which possesses significant probiotic potential, with enhanced acid/bile tolerance, attachment to porcine intestinal epithelial cells (IPEC-J2), and antimicrobial activity. The genetic characteristics of strain JDFM LP11 were explored by performing whole genome sequencing (WGS) using a PacBio system. The circular draft genome have a total length of 3,206,883 bp and a total of 3,021 coding sequences were identified. Phylogenetically, three genes, possibly related to survival and metabolic activity in the porcine host, were identified. These genes encode p60, lichenan permease IIC component, and protein TsgA, which are a putative endopeptidase, a component of the phosphotransferase system (PTS), and a major facilitator in the gut environment, respectively. Our findings suggest that understanding the functional and genetic characteristics of L. plantarum strain JDFM LP11, with its candidate genes for gut health, could provide new opportunities and insights into applications in the animal food and feed additive industries.

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September 22, 2019

How long are long tandem repeats? A challenge for current methods of whole-genome sequence assembly: The case of satellites in Caenorhabditis elegans.

Repetitive genome regions have been difficult to sequence, mainly because of the comparatively small size of the fragments used in assembly. Satellites or tandem repeats are very abundant in nematodes and offer an excellent playground to evaluate different assembly methods. Here, we compare the structure of satellites found in three different assemblies of the Caenorhabditis elegans genome: the original sequence obtained by Sanger sequencing, an assembly based on PacBio technology, and an assembly using Nanopore sequencing reads. In general, satellites were found in equivalent genomic regions, but the new long-read methods (PacBio and Nanopore) tended to result in longer assembled satellites. Important differences exist between the assemblies resulting from the two long-read technologies, such as the sizes of long satellites. Our results also suggest that the lengths of some annotated genes with internal repeats which were assembled using Sanger sequencing are likely to be incorrect.

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September 22, 2019

Repeat elements organise 3D genome structure and mediate transcription in the filamentous fungus Epichloë festucae.

Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on how genome structure and nuclear organisation influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloë festucae. We use Hi-C data to examine the three-dimensional organisation of the genome, and RNA-seq data to investigate how Epichloë genome structure contributes to the suite of transcriptional changes needed to maintain symbiotic relationships with the grass host. Our results reveal a genome in which very repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences that are almost repeat-free. In contrast to other species reported to date, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism.

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September 22, 2019

Complete genome sequence and characterization of linezolid-resistant Enterococcus faecalis clinical isolate KUB3006 carrying a cfr(B)-transposon on its chromosome and optrA-plasmid.

Linezolid (LZD) has become one of the most important antimicrobial agents for infections caused by gram-positive bacteria, including those caused by Enterococcus species. LZD-resistant (LR) genetic features include mutations in 23S rRNA/ribosomal proteins, a plasmid-borne 23S rRNA methyltransferase gene cfr, and ribosomal protection genes (optrA and poxtA). Recently, a cfr gene variant, cfr(B), was identified in a Tn6218-like transposon (Tn) in a Clostridioides difficile isolate. Here, we isolated an LR Enterococcus faecalis clinical isolate, KUB3006, from a urine specimen of a patient with urinary tract infection during hospitalization in 2017. Comparative and whole-genome analyses were performed to characterize the genetic features and overall antimicrobial resistance genes in E. faecalis isolate KUB3006. Complete genome sequencing of KUB3006 revealed that it carried cfr(B) on a chromosomal Tn6218-like element. Surprisingly, this Tn6218-like element was almost (99%) identical to that of C. difficile Ox3196, which was isolated from a human in the UK in 2012, and to that of Enterococcus faecium 5_Efcm_HA-NL, which was isolated from a human in the Netherlands in 2012. An additional oxazolidinone and phenicol resistance gene, optrA, was also identified on a plasmid. KUB3006 is sequence type (ST) 729, suggesting that it is a minor ST that has not been reported previously and is unlikely to be a high-risk E. faecalis lineage. In summary, LR E. faecalis KUB3006 possesses a notable Tn6218-like-borne cfr(B) and a plasmid-borne optrA. This finding raises further concerns regarding the potential declining effectiveness of LZD treatment in the future.

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September 22, 2019

Loss of bacitracin resistance due to a large genomic deletion among Bacillus anthracis strains.

Bacillus anthracis is a Gram-positive endospore-forming bacterial species that causes anthrax in both humans and animals. In Zambia, anthrax cases are frequently reported in both livestock and wildlife, with occasional transmission to humans, causing serious public health problems in the country. To understand the genetic diversity of B. anthracis strains in Zambia, we sequenced and compared the genomic DNA of B. anthracis strains isolated across the country. Single nucleotide polymorphisms clustered these strains into three groups. Genome sequence comparisons revealed a large deletion in strains belonging to one of the groups, possibly due to unequal crossing over between a pair of rRNA operons. The deleted genomic region included genes conferring resistance to bacitracin, and the strains with the deletion were confirmed with loss of bacitracin resistance. Similar deletions between rRNA operons were also observed in a few B. anthracis strains phylogenetically distant from Zambian strains. The structure of bacitracin resistance genes flanked by rRNA operons was conserved only in members of the Bacillus cereus group. The diversity and genomic characteristics of B. anthracis strains determined in this study would help in the development of genetic markers and treatment of anthrax in Zambia. IMPORTANCE Anthrax is caused by Bacillus anthracis, an endospore-forming soil bacterium. The genetic diversity of B. anthracis is known to be low compared with that of Bacillus species. In this study, we performed whole-genome sequencing of Zambian isolates of B. anthracis to understand the genetic diversity between closely related strains. Comparison of genomic sequences revealed that closely related strains were separated into three groups based on single nucleotide polymorphisms distributed throughout the genome. A large genomic deletion was detected in the region containing a bacitracin resistance gene cluster flanked by rRNA operons, resulting in the loss of bacitracin resistance. The structure of the deleted region, which was also conserved among species of the Bacillus cereus group, has the potential for both deletion and amplification and thus might be enabling the species to flexibly control the level of bacitracin resistance for adaptive evolution.

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September 22, 2019

Genome sequence and metabolic analysis of a fluoranthene-degrading strain Pseudomonas aeruginosa DN1.

Pseudomonas aeruginosa DN1, isolated from petroleum-contaminated soil, showed excellent degradation ability toward diverse polycyclic aromatic hydrocarbons (PAHs). Many studies have been done to improve its degradation ability. However, the molecular mechanisms of PAHs degradation in DN1 strain are unclear. In this study, the whole genome of DN1 strain was sequenced and analyzed. Its genome contains 6,641,902 bp and encodes 6,684 putative open reading frames (ORFs), which has the largest genome in almost all the comparative Pseudomonas strains. Results of gene annotation showed that this strain harbored over 100 candidate genes involved in PAHs degradation, including those encoding 25 dioxygenases, four ring-hydroxylating dioxygenases, five ring-cleaving dioxygenases, and various catabolic enzymes, transcriptional regulators, and transporters in the degradation pathways. In addition, gene knockout experiments revealed that the disruption of some key PAHs degradation genes in DN1 strain, such as catA, pcaG, pcaH, and rhdA, did not completely inhibit fluoranthene degradation, even though their degradative rate reduced to some extent. Three intermediate metabolites, including 9-hydroxyfluorene, 1-acenaphthenone, and 1, 8-naphthalic anhydride, were identified as the dominating intermediates in presence of 50 µg/mL fluoranthene as the sole carbon source according to gas chromatography mass spectrometry analysis. Taken together, the genomic and metabolic analysis indicated that the fluoranthene degradation by DN1 strain was initiated by dioxygenation at the C-1, 2-, C-2, 3-, and C-7, 8- positions. These results provide new insights into the genomic plasticity and environmental adaptation of DN1 strain.

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September 22, 2019

Characterization and genomic analyses of Pseudomonas aeruginosa podovirus TC6: establishment of genus Pa11virus.

Phages have attracted a renewed interest as alternative to chemical antibiotics. Although the number of phages is 10-fold higher than that of bacteria, the number of genomically characterized phages is far less than that of bacteria. In this study, phage TC6, a novel lytic virus of Pseudomonas aeruginosa, was isolated and characterized. TC6 consists of an icosahedral head with a diameter of approximately 54 nm and a short tail with a length of about 17 nm, which are characteristics of the family Podoviridae. TC6 can lyse 86 out of 233 clinically isolated P. aeruginosa strains, thus showing application potentials for phage therapy. The linear double-stranded genomic DNA of TC6 consisted of 49796 base pairs and was predicted to contain 71 protein-coding genes. A total of 11 TC6 structural proteins were identified by mass spectrometry. Comparative analysis revealed that the P. aeruginosa phages TC6, O4, PA11, and IME180 shared high similarity at DNA sequence and proteome levels, among which PA11 was the first phage discovered and published. Meanwhile, these phages contain 54 core genes and have very close phylogenetic relationships, which distinguish them from other known phage genera. We therefore proposed that these four phages can be classified as Pa11virus, comprising a new phage genus of Podoviridae that infects Pseudomonas spp. The results of this work promoted our understanding of phage biology, classification, and diversity.

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September 22, 2019

Therapeutic potential of a new jumbo phage that infects Vibrio coralliilyticus, a widespread coral pathogen.

Biological control using bacteriophages is a promising approach for mitigating the devastating effects of coral diseases. Several phages that infect Vibrio coralliilyticus, a widespread coral pathogen, have been isolated, suggesting that this bacterium is permissive to viral infection and is, therefore, a suitable candidate for treatment by phage therapy. In this study, we combined functional and genomic approaches to evaluate the therapeutic potential of BONAISHI, a novel V. coralliilyticus phage, which was isolated from the coral reef in Van Phong Bay (Vietnam). BONAISHI appears to be strictly lytic for several pathogenic strains of V. coralliilyticus and remains infectious over a broad range of environmental conditions. This candidate has an unusually large dsDNA genome (303 kb), with no genes that encode known toxins or implicated in lysogeny control. We identified several proteins involved in host lysis, which may offer an interesting alternative to the use of whole bacteriophages for controlling V. coralliilyticus. A preliminary therapy test showed that adding BONAISHI to an infected culture of Symbiodinium sp. cells reduced the impact of V. coralliilyticus on Symbiodinium sp. photosynthetic activity. This study showed that BONAISHI is able to mitigate V. coralliilyticus infections, making it a good candidate for phage therapy for coral disease.

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September 22, 2019

A complete Cannabis chromosome assembly and adaptive admixture for elevated cannabidiol (CBD) content

Cannabis has been cultivated for millennia with distinct cultivars providing either fiber and grain or tetrahydrocannabinol. Recent demand for cannabidiol rather than tetrahydrocannabinol has favored the breeding of admixed cultivars with extremely high cannabidiol content. Despite several draft Cannabis genomes, the genomic structure of cannabinoid synthase loci has remained elusive. A genetic map derived from a tetrahydrocannabinol/cannabidiol segregating population and a complete chromosome assembly from a high-cannabidiol cultivar together resolve the linkage of cannabidiolic and tetrahydrocannabinolic acid synthase gene clusters which are associated with transposable elements. High-cannabidiol cultivars appear to have been generated by integrating hemp-type cannabidiolic acid synthase gene clusters into a background of marijuana-type cannabis. Quantitative trait locus mapping suggests that overall drug potency, however, is associated with other genomic regions needing additional study.

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September 22, 2019

SKA: Split Kmer Analysis Toolkit for Bacterial Genomic Epidemiology

Genome sequencing is revolutionising infectious disease epidemiology, providing a huge step forward in sensitivity and specificity over more traditional molecular typing techniques. However, the complexity of genome data often means that its analysis and interpretation requires high-performance compute infrastructure and dedicated bioinformatics support. Furthermore, current methods have limitations that can differ between analyses and are often opaque to the user, and their reliance on multiple external dependencies makes reproducibility difficult. Here I introduce SKA, a toolkit for analysis of genome sequence data from closely-related, small, haploid genomes. SKA uses split kmers to rapidly identify variation between genome sequences, making it possible to analyse hundreds of genomes on a standard home computer. Tests on publicly available simulated and real-life data show that SKA is both faster and more efficient than the gold standard methods used today while retaining similar levels of accuracy for epidemiological purposes. SKA can take raw read data or genome assemblies as input and calculate pairwise distances, create single linkage clusters and align genomes to a reference genome or using a reference-free approach. SKA requires few decisions to be made by the user, which, along with its computational efficiency, allows genome analysis to become accessible to those with only basic bioinformatics training. The limitations of SKA are also far more transparent than for current approaches, and future improvements to mitigate these limitations are possible. Overall, SKA is a powerful addition to the armoury of the genomic epidemiologist. SKA source code is available from Github (https://github.com/simonrharris/SKA).

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September 22, 2019

Update on Tetracycline Susceptibility of Pediococcus acidilactici Based on Strains Isolated from Swiss Cheese and Whey.

Bacterial strains used as starter cultures in the production of fermented foods may act as reservoirs for antibiotic resistance (AbR) genes. To avoid the introduction of such genes into the food chain, the presence of acquired AbR in bacterial strains added to food must be tested. Standard protocols and microbiological cut-off values have been defined to provide practitioners with a basis for evaluating whether their bacterial isolates harbor an acquired resistance to a given antibiotic. Here, we tested the AbR of 24 strains of Pediococcus acidilactici by using the standard protocol and microbiological cut-off values recommended by the European Food Safety Authority. Phenotypic data were complemented by searching for known AbR genes using an in silico analysis of whole genomes. The majority (54.2%) of the strains were able to grow at a tetracycline concentration above the defined cut-off, even though only one strain carried a known tetracycline resistance gene, tetM. The same strain also carried the AbR gene of an erythromycin resistance methylase, ermA, and displayed resistance toward clindamycin and erythromycin. Our results bolster the scarce data on the sensitivity of P. acidilactici to tetracycline and suggest that the microbiological cut-off recommended by the European Food Safety Authority for this antibiotic should be amended.

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September 22, 2019

Eco-friendly Management of Karnal Bunt (Neovossia indica) of Wheat

Karnal bunt incited by Neovossia indica is one of the most important disease of wheat crop. To develop an eco-friendly management practice against Karnal bunt of wheat, integration of fungicidal seed treatment with foliar sprays of phytoextracts, bio-control agent and fungicide revealed. Uses of Thiram 75DS or Kavach 75WP @2g/Kg, Dithane M-45 or Captan [email protected]/Kg, Vitavax [email protected]/Kg, Tilt 25EC or Raxil 2DS@1mL/Kg or Pseudomonas fluorescens@5 mL/Kg or Trichoderma viride (Ecoderma) or T. harzianum@5 mL/Kg seed treatment for eliminating primary inoculum (teliospores). Seed soaking in Lantana (L. camara) or Eucalyptus (E. globulus) or Akh (Calotropis procera) or Kali basuti (Eupatorium adenophorum) @ 250 mL/L for 60 min and dry in shad are effective in eradicating the seed infection also. Application foliar spray of Baycor 25WP or Bavistin 50WP or F-100 or Moximate [email protected]/Kg, Tilt 25EC or Folicur 25EC or Contaf 25EC@1mL/Kg at boot leaf stage and 50% emergence flowering heads against the secondary air-borne inoculum (Allantoides sporidia). This is concerning integration of fungicide seed treatment with foliar spray of bio- control agent and phyto-extract. It is cheaper and eco-friendly practice for the control of Karnal bunt of wheat.

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September 22, 2019

Physiological genomics of dietary adaptation in a marine herbivorous fish

Adopting a new diet is a significant evolutionary change and can profoundly affect an animaltextquoterights physiology, biochemistry, ecology, and its genome. To study this evolutionary transition, we investigated the physiology and genomics of digestion of a derived herbivorous fish, the monkeyface prickleback (Cebidichthys violaceus). We sequenced and assembled its genome and digestive transcriptome and revealed the molecular changes related to important dietary enzymes, finding abundant evidence for adaptation at the molecular level. In this species, two gene families experienced expansion in copy number and adaptive amino acid substitutions. These families, amylase, and bile salt activated lipase, are involved digestion of carbohydrates and lipids, respectively. Both show elevated levels of gene expression and increased enzyme activity. Because carbohydrates are abundant in the pricklebacktextquoterights diet and lipids are rare, these findings suggest that such dietary specialization involves both exploiting abundant resources and scavenging rare ones, especially essential nutrients, like essential fatty acids.

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September 22, 2019

Antimicrobial resistance profile of mcr-1 positive clinical isolates of Escherichia coli in China From 2013 to 2016.

Multidrug-resistant (MDR) Escherichia coli poses a great challenge for public health in recent decades. Polymyxins have been reconsidered as a valuable therapeutic option for the treatment of infections caused by MDR E. coli. A plasmid-encoded colistin resistance gene mcr-1 encoding phosphoethanolamine transferase has been recently described in Enterobacteriaceae. In this study, a total of 123 E. coli isolates obtained from patients with diarrheal diseases in China were used for the genetic analysis of colistin resistance in clinical isolates. Antimicrobial resistance profile of polymyxin B (PB) and 11 commonly used antimicrobial agents were determined. Among the 123 E. coli isolates, 9 isolates (7.3%) were resistant to PB and PCR screening showed that seven (5.7%) isolates carried the mcr-1 gene. A hybrid sequencing analysis using single-molecule, real-time (SMRT) sequencing and Illumina sequencing was then performed to resolve the genomes of the seven mcr-1 positive isolates. These seven isolates harbored multiple plasmids and are MDR, with six isolates carrying one mcr-1 positive plasmid and one isolate (14EC033) carrying two mcr-1 positive plasmids. These eight mcr-1 positive plasmids belonged to the IncX4, IncI2, and IncP1 types. In addition, the mcr-1 gene was the solo antibiotic resistance gene identified in the mcr-1 positive plasmids, while the rest of the antibiotic resistance genes were mostly clustered into one or two plasmids. Interestingly, one mcr-1 positive isolate (14EC047) was susceptible to PB, and we showed that the activity of MCR-1-mediated colistin resistance was not phenotypically expressed in 14EC047 host strain. Furthermore, three isolates exhibited resistance to PB but did not carry previously reported mcr-related genes. Multilocus sequence typing (MLST) showed that these mcr-1 positive E. coli isolates belonged to five different STs, and three isolates belonged to ST301 which carried multiple virulence factors related to diarrhea. Additionally, the mcr-1 positive isolates were all susceptible to imipenem (IMP), suggesting that IMP could be used to treat infection caused by mcr-1 positive E. coli isolates. Collectively, this study showed a high occurrence of mcr-1 positive plasmids in patients with diarrheal diseases of Guangzhou in China and the abolishment of the MCR-1 mediated colistin resistance in one E. coli isolate.

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