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Auto Tag: Transposase

September 22, 2019  |  

Identification of the KPC plasmid pCT-KPC334: New insights on the evolutionary pathway of epidemic plasmids harboring fosA3-blaKPC-2 genes.

A novel, non-conjugative plasmid pKP1034 isolated from a fosfomycin-resistant, carbapenemase-producing Klebsiella pneumonia strain KP1034 was recently reported to carry fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12 and rmtB genes, and was hypothesized to evolve from several recombination events of two closely related plasmids, pHN7A8 and pKPC-LK30 [1]. In this study, a plasmid pCT-KPC334 carrying fosA3, blaKPC-2, blaCTX-M-65, blaSHV-12, blaTEM-1, and rmtB genes was identified, providing evidence on the evolutionary pathway of plasmids harboring fosA3-blaKPC-2 genes.

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September 22, 2019  |  

Dissemination and persistence of extended-spectrum cephalosporin-resistance encoding IncI1-blaCTXM-1 plasmid among Escherichia coli in pigs.

This study investigated the ecology, epidemiology and plasmid characteristics of extended-spectrum cephalosporin (ESC)-resistant E. coli in healthy pigs over a period of 4 years (2013-2016) following the withdrawal of ESCs. High carriage rates of ESC-resistant E. coli were demonstrated in 2013 (86.6%) and 2014 (83.3%), compared to 2015 (22%) and 2016 (8.5%). ESC resistance identified among E. coli isolates was attributed to the carriage of an IncI1 ST-3 plasmid (pCTXM1-MU2) encoding blaCTXM-1. Genomic characterisation of selected E. coli isolates (n?=?61) identified plasmid movement into multiple commensal E. coli (n?=?22 STs). Major STs included ST10, ST5440, ST453, ST2514 and ST23. A subset of the isolates belong to the atypical enteropathogenic E. coli (aEPEC) pathotype that harboured multiple LEE pathogenic islands. pCTXM1-MU2 was similar (99% nt identity) to IncI1-ST3 plasmids reported from Europe, encoded resistance to aminoglycosides, sulphonamides and trimethoprim, and carried colicin Ib. pCTXM1-MU2 appears to be highly stable and readily transferable. This study demonstrates that ESC resistance may persist for a protracted period following removal of direct selection pressure, resulting in the emergence of ESC-resistance in both commensal E. coli and aEPEC isolates of potential significance to human and animal health.

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September 22, 2019  |  

Bias in resistance gene prediction due to repeat masking

Several recently published Brassicaceae genome annotations show strong differences in resistance (R)-gene content. We believe that this is caused by different approaches to repeat masking. Here we show that some of the repeats stored in public databases used for repeat masking carry pieces of predicted R-gene-related domains, and demonstrate that at least some of the variance in R-gene content in recent genome annotations is caused by using these repeats for repeat masking. We also show that other classes of genes are less affected by this phenomenon, and estimate a false positive rate of R genes (0 to 4.6%) that are in reality transposons carrying the R-gene domains. These results may partially explain why there has been a decrease in published novel R genes in recent years, which has implications for plant breeding, especially in the face of pathogens changing as a response to climate change.

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September 22, 2019  |  

Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase.

Here we present APOBEC-coupled epigenetic sequencing (ACE-seq), a bisulfite-free method for localizing 5-hydroxymethylcytosine (5hmC) at single-base resolution with low DNA input. The method builds on the observation that AID/APOBEC family DNA deaminase enzymes can potently discriminate between cytosine modification states and exploits the non-destructive nature of enzymatic, rather than chemical, deamination. ACE-seq yielded high-confidence 5hmC profiles with at least 1,000-fold less DNA input than conventional methods. Applying ACE-seq to generate a base-resolution map of 5hmC in tissue-derived cortical excitatory neurons, we found that 5hmC was almost entirely confined to CG dinucleotides. The whole-genome map permitted cytosine, 5-methylcytosine (5mC) and 5hmC to be parsed and revealed genomic features that diverged from global patterns, including enhancers and imprinting control regions with high and low 5hmC/5mC ratios, respectively. Enzymatic deamination overcomes many challenges posed by bisulfite-based methods, thus expanding the scope of epigenome profiling to include scarce samples and opening new lines of inquiry regarding the role of cytosine modifications in genome biology.

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September 22, 2019  |  

Convergent evolution of complex genomic rearrangements in two fungal meiotic drive elements.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3, found in the filamentous ascomycete Neurospora intermedia. Using long-read sequencing, we generated the first complete and well-annotated genome assemblies of large, highly diverged, non-recombining regions associated with meiotic drive elements. Phylogenetic analysis shows that, even though Sk-2 and Sk-3 are located in the same chromosomal region, they do not form sister clades, suggesting independent origins or at least a long evolutionary separation. We conclude that they have in a convergent manner accumulated similar patterns of tandem inversions and dense repeat clusters, presumably in response to similar needs to create linkage between genes causing drive and resistance.

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September 22, 2019  |  

Genomic analysis of the Phalaenopsis pathogen Dickeya sp. PA1, representing the emerging species Dickeya fangzhongdai.

Dickeya sp. strain PA1 is the causal agent of bacterial soft rot in Phalaenopsis, an important indoor orchid in China. PA1 and a few other strains were grouped into a novel species, Dickeya fangzhongdai, and only the orchid-associated strains have been shown to cause soft rot symptoms.We constructed the complete PA1 genome sequence and used comparative genomics to explore the differences in genomic features between D. fangzhongdai and other Dickeya species.PA1 has a 4,979,223-bp circular genome with 4269 predicted protein-coding genes. D. fangzhongdai was phylogenetically similar to Dickeya solani and Dickeya dadantii. The type I to type VI secretion systems (T1SS-T6SS), except for the stt-type T2SS, were identified in D. fangzhongdai. The three phylogenetically similar species varied significantly in terms of their T5SSs and T6SSs, as did the different D. fangzhongdai strains. Genomic island (GI) prediction and synteny analysis (compared to D. fangzhongdai strains) of PA1 also indicated the presence of T5SSs and T6SSs in strain-specific regions. Two typical CRISPR arrays were identified in D. fangzhongdai and in most other Dickeya species, except for D. solani. CRISPR-1 was present in all of these Dickeya species, while the presence of CRISPR-2 varied due to species differentiation. A large polyketide/nonribosomal peptide (PK/NRP) cluster, similar to the zeamine biosynthetic gene cluster in Dickeya zeae rice strains, was discovered in D. fangzhongdai and D. solani. The D. fangzhongdai and D. solani strains might recently have acquired this gene cluster by horizontal gene transfer (HGT).Orchid-associated strains are the typical members of D. fangzhongdai. Genomic analysis of PA1 suggested that this strain presents the genomic characteristics of this novel species. Considering the absence of the stt-type T2SS, the presence of CRISPR loci and the zeamine biosynthetic gene cluster, D. fangzhongdai is likely a transitional form between D. dadantii and D. solani. This is supported by the later acquisition of the zeamine cluster and the loss of CRISPR arrays by D. solani. Comparisons of phylogenetic positions and virulence determinants could be helpful for the effective quarantine and control of this emerging species.

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September 22, 2019  |  

The landscape of repetitive elements in the refined genome of chilli anthracnose fungus Colletotrichum truncatum.

The ascomycete fungus Colletotrichum truncatum is a major phytopathogen with a broad host range which causes anthracnose disease of chilli. The genome sequencing of this fungus led to the discovery of functional categories of genes that may play important roles in fungal pathogenicity. However, the presence of gaps in C. truncatum draft assembly prevented the accurate prediction of repetitive elements, which are the key players to determine the genome architecture and drive evolution and host adaptation. We re-sequenced its genome using single-molecule real-time (SMRT) sequencing technology to obtain a refined assembly with lesser and smaller gaps and ambiguities. This enabled us to study its genome architecture by characterising the repetitive sequences like transposable elements (TEs) and simple sequence repeats (SSRs), which constituted 4.9 and 0.38% of the assembled genome, respectively. The comparative analysis among different Colletotrichum species revealed the extensive repeat rich regions, dominated by Gypsy superfamily of long terminal repeats (LTRs), and the differential composition of SSRs in their genomes. Our study revealed a recent burst of LTR amplification in C. truncatum, C. higginsianum, and C. scovillei. TEs in C. truncatum were significantly associated with secretome, effectors and genes in secondary metabolism clusters. Some of the TE families in C. truncatum showed cytosine to thymine transitions indicative of repeat-induced point mutation (RIP). C. orbiculare and C. graminicola showed strong signatures of RIP across their genomes and “two-speed” genomes with extensive AT-rich and gene-sparse regions. Comparative genomic analyses of Colletotrichum species provided an insight into the species-specific SSR profiles. The SSRs in the coding and non-coding regions of the genome revealed the composition of trinucleotide repeat motifs in exons with potential to alter the translated protein structure through amino acid repeats. This is the first genome-wide study of TEs and SSRs in C. truncatum and their comparative analysis with six other Colletotrichum species, which would serve as a useful resource for future research to get insights into the potential role of TEs in genome expansion and evolution of Colletotrichum fungi and for development of SSR-based molecular markers for population genomic studies.

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September 22, 2019  |  

Bacillus wiedmannii biovar thuringiensis: A specialized mosquitocidal pathogen with plasmids from diverse origins.

Bacillus cereus sensu lato also known as B. cereus group is composed of an ecologically diverse bacterial group with an increasing number of related species, some of which are medically or agriculturally important. Numerous e?orts have been undertaken to allow presumptive di?erentiation of B. cereus group species from one another. FCC41 is a Bacillus sp. strain toxic against mosquito species like Aedes aegypti, Aedes (Ochlerotatus) albifasciatus, Culex pipiens, Culex quinquefasciatus, and Culex apicinus, some of them responsible for the transmission of vector-borne diseases. Here, we report the complete genome sequence of FCC41 strain, which consists of one circular chromosome and eight circular plasmids ranging in size from 8 to 490?kb. This strain harbors six crystal protein genes, including cry24Ca, two cry4-like and two cry52-like, a cry41-like parasporin gene and multiple virulence factors. The phylogenetic analysis of the whole-genome sequence of this strain with molecular approaches places this strain into the Bacillus wiedmannii cluster. However, according with phenotypical characteristics such as the mosquitocidal activity due to the presence of Cry proteins found in the parasporal body and cry genes encoded in plasmids of different sizes, indicate that this strain could be renamed as B. wiedmannii biovar thuringiensis strain FCC41.

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September 22, 2019  |  

Distribution of the pco gene cluster and associated genetic determinants among swine Escherichia coli from a controlled feeding trial.

Copper is used as an alternative to antibiotics for growth promotion and disease prevention. However, bacteria developed tolerance mechanisms for elevated copper concentrations, including those encoded by the pco operon in Gram-negative bacteria. Using cohorts of weaned piglets, this study showed that the supplementation of feed with copper concentrations as used in the field did not result in a significant short-term increase in the proportion of pco-positive fecal Escherichia coli. The pco and sil (silver resistance) operons were found concurrently in all screened isolates, and whole-genome sequencing showed that they were distributed among a diversity of unrelated E. coli strains. The presence of pco/sil in E. coli was not associated with elevated copper minimal inhibitory concentrations (MICs) under a variety of conditions. As found in previous studies, the pco/sil operons were part of a Tn7-like structure found both on the chromosome or on plasmids in the E. coli strains investigated. Transfer of a pco/sil IncHI2 plasmid from E. coli to Salmonellaenterica resulted in elevated copper MICs in the latter. Escherichia coli may represent a reservoir of pco/sil genes transferable to other organisms such as S. enterica, for which it may represent an advantage in the presence of copper. This, in turn, has the potential for co-selection of resistance to antibiotics.

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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  |  

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  |  

The Butanol Producing Microbe Clostridium beijerinckii NCIMB 14988 Manipulated Using Forward and Reverse Genetic Tools.

The solventogenic anaerobe Clostridium beijerinckii has potential for use in the sustainable bioconversion of plant-derived carbohydrates into solvents, such as butanol or acetone. However, relatively few strains have been extensively characterised either at the genomic level or through exemplification of a complete genetic toolkit. To remedy this situation, a new strain of C. beijerinckii, NCIMB 14988, is selected from among a total of 55 new clostridial isolates capable of growth on hexose and pentose sugars. Chosen on the basis of its favorable properties, the complete genome sequence of NCIMB 14988 is determined and a high-efficiency plasmid transformation protocol devised. The developed DNA transfer procedure allowed demonstration in NCIMB 14988 of the forward and reverse genetic techniques of transposon mutagenesis and gene knockout, respectively. The latter is accomplished through the successful deployment of both group II intron retargeting (ClosTron) and allelic exchange. In addition to gene inactivation, the developed allelic exchange procedure is used to create point mutations in the chromosome, allowing for the effect of amino acid changes in enzymes involved in primary metabolism to be characterized. ClosTron mediated disruption of the currently unannotated non-coding region between genes LF65_05915 and LF65_05920 is found to result in a non-sporulating phenotype.© 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

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September 22, 2019  |  

Comparative genomics of Staphylococcus reveals determinants of speciation and diversification of antimicrobial defense.

The bacterial genus Staphylococcus comprises diverse species with most being described as colonizers of human and animal skin. A relational analysis of features that discriminate its species and contribute to niche adaptation and survival remains to be fully described. In this study, an interspecies, whole-genome comparative analysis of 21 Staphylococcus species was performed based on their orthologues. Three well-defined multi-species groups were identified: group A (including aureus/epidermidis); group B (including saprophyticus/xylosus) and group C (including pseudintermedius/delphini). The machine learning algorithm Random Forest was applied to prioritize orthologs that drive formation of the Staphylococcus species groups A-C. Orthologues driving staphylococcal intrageneric diversity comprised regulatory, metabolic and antimicrobial resistance proteins. Notably, the BraSR (NsaRS) two-component system (TCS) and its associated BraDE transporters that regulate antimicrobial resistance showed limited distribution in the genus and their presence was most closely associated with a subset of Staphylococcus species dominated by those that colonize human skin. Divergence of BraSR and GraSR antimicrobial peptide survival TCS and their associated transporters was observed across the staphylococci, likely reflecting niche specific evolution of these TCS/transporters and their specificities for AMPs. Experimental evolution, with selection for resistance to the lantibiotic nisin, revealed multiple routes to resistance and differences in the selection outcomes of the BraSR-positive species S. hominis and S. aureus. Selection supported a role for GraSR in nisin survival responses of the BraSR-negative species S. saprophyticus. Our study reveals diversification of antimicrobial-sensing TCS across the staphylococci and hints at differential relationships between GraSR and BraSR in those species positive for both TCS.

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September 22, 2019  |  

Whole-Genome Analysis of an Extensively Drug-Resistant Acinetobacter baumannii Strain XDR-BJ83: Insights into the Mechanisms of Resistance of an ST368 Strain from a Tertiary Care Hospital in China.

Acinetobacter baumannii is an important pathogen of nosocomial infections. Nosocomial outbreaks caused by antibiotic-resistant A. baumannii remain a significant challenge. Understanding the antibiotic resistance mechanism of A. baumannii is critical for clinical treatment. The purpose of this study was to determine the whole-genome sequence (WGS) of an extensively drug-resistant (XDR) A. baumannii strain, XDR-BJ83, which was associated with a nosocomial outbreak in a tertiary care hospital of China, and to investigate the antibiotic resistance mechanism of this strain. The WGS of XDR-BJ83 was performed using single-molecule real-time sequencing. The complete genome of XDR-BJ83 consisted of a 4,011,552-bp chromosome and a 69,069-bp plasmid. The sequence type of XDR-BJ83 was ST368, which belongs to clonal complex 92 (CC92). The chromosome of XDR-BJ83 carried multiple antibiotic resistance genes, antibiotic efflux pump genes, and mobile genetic elements, including insertion sequences, transposons, integrons, and resistance islands. The plasmid of XDR-BJ83 (pBJ83) was a conjugative plasmid carrying type IV secretion system. These results indicate that the presence of multiple antibiotic resistance genes, efflux pumps, and mobile genetic elements is likely associated with resistance to various antibiotics in XDR-BJ83.

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September 22, 2019  |  

Thermosipho spp. immune system differences affect variation in genome size and geographical distributions.

Thermosipho species inhabit thermal environments such as marine hydrothermal vents, petroleum reservoirs, and terrestrial hot springs. A 16S rRNA phylogeny of available Thermosipho spp. sequences suggested habitat specialists adapted to living in hydrothermal vents only, and habitat generalists inhabiting oil reservoirs, hydrothermal vents, and hotsprings. Comparative genomics of 15 Thermosipho genomes separated them into three distinct species with different habitat distributions: The widely distributed T. africanus and the more specialized, T. melanesiensis and T. affectus. Moreover, the species can be differentiated on the basis of genome size (GS), genome content, and immune system composition. For instance, the T. africanus genomes are largest and contained the most carbohydrate metabolism genes, which could explain why these isolates were obtained from ecologically more divergent habitats. Nonetheless, all the Thermosipho genomes, like other Thermotogae genomes, show evidence of genome streamlining. GS differences between the species could further be correlated to differences in defense capacities against foreign DNA, which influence recombination via HGT. The smallest genomes are found in T. affectus that contain both CRISPR-cas Type I and III systems, but no RM system genes. We suggest that this has caused these genomes to be almost devoid of mobile elements, contrasting the two other species genomes that contain a higher abundance of mobile elements combined with different immune system configurations. Taken together, the comparative genomic analyses of Thermosipho spp. revealed genetic variation allowing habitat differentiation within the genus as well as differentiation with respect to invading mobile DNA.

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