scholarly journals Illuminating the transposon insertion landscape in plants using Cas9-targeted Nanopore sequencing and a novel pipeline

2021 ◽  
Author(s):  
Ilya Kirov ◽  
Pavel Merkulov ◽  
Sofya Gvaramiya ◽  
Roman Komakhin ◽  
Murad Omarov ◽  
...  
Keyword(s):  
Aegilops Tauschii ◽  
Nanopore Sequencing ◽  
Wild Type ◽  
Plant Adaptation ◽  
Genome Coverage ◽  
Large Genome ◽  
Plant Genomes ◽  
Transposon Insertion ◽  
Gypsy Retrotransposon ◽  
Ddm1 Mutant

Transposable elements (TEs), which occupy significant portions of most plant genomes, are a major source of genomic novelty, contributing to plant adaptation, speciation and new cultivar production. The often large, complex genomes of plants make identifying TE insertions from short reads challenging, while whole-genome sequencing remains expensive. To expand the toolbox for TE identification in plants, we used the recently developed Cas9-targeted Nanopore sequencing (CANS) approach. Additionally, as no current bioinformatics tools automatically detect TE insertions after CANS, we developed NanoCasTE, a novel pipeline for target TE insertion discovery. We performed CANS of three copies of EVD retrotransposons in wild-type Arabidopsis thaliana and obtained up to 40x coverage of the targets after only a few hours of sequencing on a MinION sequencer. To estimate the ability to detect new TE insertions, we exploited the A. thaliana ddm1 mutant, which has elevated TE activity. Using CANS, we detected 84% of these insertions in ddm1 after generating only 4420 Nanopore reads (0.2x genome coverage), and also unambiguously identified their locations, demonstrating the method's sensitivity. CANS of pooled (~50 plants) ddm1 plants captured >800 EVD insertions, especially in centromeric regions. CANS also identified insertions of a Ty3/Gypsy retrotransposon in the genomes of two Aegilops tauschii plants, a species with a large genome.

scholarly journals OmpR and LeuO Positively Regulate the Salmonella enterica Serovar Typhi ompS2 Porin Gene

2004 ◽  
Vol 186 (10) ◽  
pp. 2909-2920 ◽  
Author(s):  
Marcos Fernández-Mora ◽  
José Luis Puente ◽  
Edmundo Calva
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Phosphorylation Site ◽  
Regulatory Region ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Upstream Regulatory Region ◽  
Wild Type ◽  
Transposon Insertion

ABSTRACT The Salmonella enterica serovar Typhi ompS2 gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily. It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for S. enterica serovar Typhi OmpS1, Escherichia coli OmpN, and Klebsiella pneumoniae OmpK37 quiescent porins. A region of 316 bp, between nucleotides −413 and −97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in ompS2 expression in an S. enterica serovar Typhi wild-type strain. This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory ompB (ompR envZ) operon. Furthermore, ompS2 expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site. Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of ompS2 expression. Augmented expression of ompS2 was also obtained upon addition of cloned leuO to the wild-type strain, but not in an ompR isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR. Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5′ upstream regulatory region. Thus, the OmpR and LeuO regulators positively regulate ompS2.

scholarly journals AcrAB Multidrug Efflux Pump Is Associated with Reduced Levels of Susceptibility to Tigecycline (GAR-936) in Proteus mirabilis

2003 ◽  
Vol 47 (2) ◽  
pp. 665-669 ◽  
Author(s):  
Melissa A. Visalli ◽  
Ellen Murphy ◽  
Steven J. Projan ◽  
Patricia A. Bradford
Keyword(s):  
Proteus Mirabilis ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transposon Insertion ◽  
Spectrum Activity ◽  
Insertion Mutants ◽  
Broad Spectrum Activity ◽  
Increased Susceptibility

ABSTRACT Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the Proteeae. A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P. mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in P. mirabilis.

scholarly journals Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k

2012 ◽  
Vol 93 (2) ◽  
pp. 389-399 ◽  
Author(s):  
Lopamudra Giri ◽  
Michael G. Feiss ◽  
Bryony C. Bonning ◽  
David W. Murhammer
Keyword(s):  
Large Scale ◽  
Serial Passage ◽  
Autographa Californica ◽  
Wild Type ◽  
Systematic Method ◽  
Defective Interfering Particles ◽  
Extracellular Virus ◽  
Transposon Insertion ◽  
Target Sites ◽  
Insertion Sites

Accumulation of baculovirus defective interfering particle (DIP) and few polyhedra (FP) mutants is a major limitation to continuous large-scale baculovirus production in insect-cell culture. Although overcoming these mutations would result in a cheaper platform for producing baculovirus biopesticides, little is known regarding the mechanism of FP and DIP formation. This issue was addressed by comparing DIP production of wild-type (WT) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with that of a recombinant AcMNPV (denoted Ac-FPm) containing a modified fp25k gene with altered transposon insertion sites that prevented transposon-mediated production of the FP phenotype. In addition to a reduction in the incidence of the FP phenotype, DIP formation was delayed on passaging of Ac-FPm compared with WT AcMNPV. Specifically, the yield of DIP DNA in Ac-FPm was significantly lower than in WT AcMNPV up to passage 16, thereby demonstrating that modifying the transposon insertion sites increases the genomic stability of AcMNPV. A critical component of this investigation was the optimization of a systematic method based on the use of pulsed-field gel electrophoresis (PFGE) to characterize extracellular virus DNA. Specifically, PFGE was used to detect defective genomes, determine defective genome sizes and quantify the amount of defective genome within a heterogeneous genome population of passaged virus.

scholarly journals STY1 and STY2 promote the formation of apical tissues during Arabidopsis gynoecium development

Development ◽  
2002 ◽  
Vol 129 (20) ◽  
pp. 4707-4717 ◽  
Author(s):  
Sandra Kuusk ◽  
Joel J. Sohlberg ◽  
Jeff A. Long ◽  
Ingela Fridborg ◽  
Eva Sundberg
Keyword(s):  
Double Mutant ◽  
Ectopic Expression ◽  
Ring Finger ◽  
Regional Differentiation ◽  
35S Promoter ◽  
Insertion Mutation ◽  
Wild Type ◽  
Transposon Insertion ◽  
Visible Effect ◽  
Gynoecium Development

Gynoecium ontogenesis in Arabidopsis is accomplished by the co-ordinated activity of genes that control patterning and the regional differentiation of tissues, and ultimately results in the formation of a basal ovary, a short style and an apical stigma. A transposon insertion in the STYLISH1 (STY1) gene results in gynoecia with aberrant style morphology, while an insertion mutation in the closely related STYLISH2 (STY2) gene has no visible effect on gynoecium development. However, sty1-1 sty2-1 double mutant plants exhibit an enhanced sty1-1 mutant phenotype and are characterized by a further reduction in the amount of stylar and stigmatic tissues and decreased proliferation of stylar xylem. These data imply that STY1 and STY2 are partially redundant and that both genes promote style and stigma formation and influence vascular development during Arabidopsis gynoecium development. Consistently, STY1 and STY2 are expressed in the apical parts of the developing gynoecium and ectopic expression of either STY1 or STY2 driven by the CaMV 35S promoter is sufficient to transform valve cells into style cells. STY1::GUS and STY2::GUS activity is detected in many other organs as well as the gynoecium, suggesting that STY1 and STY2 may have additional functions. This is supported by the sty1-1 sty2-1 double mutants producing rosette and cauline leaves with a higher degree of serration than wild-type leaves. STY1 and STY2 are members of a small gene family, and encode proteins with a RING finger-like motif. Double mutant analyses indicate that STY1 genetically interacts with SPATULA and possibly also with CRABS CLAW.

scholarly journals Multiple Elements Controlling Adherence of Enterohemorrhagic Escherichia coli O157:H7 to HeLa Cells

2003 ◽  
Vol 71 (9) ◽  
pp. 4985-4995 ◽  
Author(s):  
Alfredo G. Torres ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Hela Cells ◽  
Culture Conditions ◽  
Enterohemorrhagic Escherichia Coli ◽  
Differentially Expressed ◽  
Wild Type ◽  
Transposon Insertion ◽  
Reduced Adherence ◽  
Insertion Mutants

ABSTRACT Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

scholarly journals Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Alexander G. Shaw ◽  
Manasi Majumdar ◽  
Catherine Troman ◽  
Áine O’Toole ◽  
Blossom Benny ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Environmental Samples ◽  
Direct Detection ◽  
Nanopore Sequencing ◽  
Wild Type ◽  
Consensus Sequences ◽  
Real Time Analysis ◽  
Detection And Identification ◽  
Culture Algorithm

ABSTRACT Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

scholarly journals Capsule Production and Glucose Metabolism Dictate Fitness duringSerratia marcescensBacteremia

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Mark T. Anderson ◽  
Lindsay A. Mitchell ◽  
Lili Zhao ◽  
Harry L. T. Mobley
Keyword(s):  
Glucose Metabolism ◽  
Serratia Marcescens ◽  
Opportunistic Infections ◽  
Opportunistic Pathogen ◽  
Extracellular Polysaccharides ◽  
Mammalian Host ◽  
Wild Type ◽  
Transposon Insertion ◽  
Capsule Production ◽  
Tract Infections

ABSTRACTSerratia marcescensis an opportunistic pathogen that causes a range of human infections, including bacteremia, keratitis, wound infections, and urinary tract infections. Compared to other members of theEnterobacteriaceaefamily, the genetic factors that facilitateSerratiaproliferation within the mammalian host are less well defined. Anin vivoscreen of transposon insertion mutants identified 212S. marcescensfitness genes that contribute to bacterial survival in a murine model of bloodstream infection. Among those identified, 11 genes were located within an 18-gene cluster encoding predicted extracellular polysaccharide biosynthesis proteins. A mutation in thewzxgene contained within this locus conferred a loss of fitness in competition infections with the wild-type strain and a reduction in extracellular uronic acids correlating with capsule loss. A second gene,pgm, encoding a phosphoglucomutase exhibited similar capsule-deficient phenotypes, linking central glucose metabolism with capsule production and fitness ofSerratiaduring mammalian infection. Further evidence of the importance of central metabolism was obtained with apfkAglycolytic mutant that demonstrated reduced replication in human serum and during murine infection. An MgtB magnesium transporter homolog was also among the fitness factors identified, and anS. marcescens mgtBmutant exhibited decreased growth in defined medium containing low concentrations of magnesium and was outcompeted ~10-fold by wild-type bacteria in mice. Together, these newly identified genes provide a more complete understanding of the specific requirements forS. marcescenssurvival in the mammalian host and provide a framework for further investigation of the means by whichS. marcescenscauses opportunistic infections.IMPORTANCESerratia marcescensis a remarkably prolific organism that replicates in diverse environments, including as an opportunistic pathogen in human bacteremia. The genetic requirements forS. marcescenssurvival in the mammalian bloodstream were defined in this work by transposon insertion sequencing. In total, 212 genes that contribute to bacterial fitness were identified. When sorted via biological function, two of the major fitness categories identified herein were genes encoding capsule polysaccharide biogenesis functions and genes involved in glucose utilization. Further investigation determined that certain glucose metabolism fitness genes are also important for the generation of extracellular polysaccharides. Together, these results identify critical biological processes that allowS. marcescensto colonize the mammalian bloodstream.

scholarly journals Nanopore Sequencing Comes to Plant Genomes

2017 ◽  
Vol 29 (11) ◽  
pp. 2677-2678
Author(s):  
Nancy R. Hofmann
Keyword(s):  
Nanopore Sequencing ◽  
Plant Genomes

scholarly journals Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 774
Author(s):  
Virginio Cepas ◽  
Victoria Ballén ◽  
Yaiza Gabasa ◽  
Miriam Ramírez ◽  
Yuly López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
De Novo ◽  
Wild Type ◽  
Planktonic Cells ◽  
E Coli ◽  
Qrt Pcr ◽  
Transposon Insertion ◽  
De Novo Purine Biosynthesis

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

scholarly journals Identification of Xylella fastidiosa Antivirulence Genes: Hemagglutinin Adhesins Contribute to X. fastidiosa Biofilm Maturation and Colonization and Attenuate Virulence

2005 ◽  
Vol 18 (8) ◽  
pp. 856-868 ◽  
Author(s):  
Magalie R. Guilhabert ◽  
Bruce C. Kirkpatrick
Keyword(s):  
Xylella Fastidiosa ◽  
Plant Diseases ◽  
Plant Interactions ◽  
Wild Type ◽  
In Planta ◽  
Transposon Tn5 ◽  
Transposon Insertion ◽  
Transposon Mutants ◽  
Major Impediment

Xylella fastidosa, a gram-negative, xylem-limited bacterium, is the causal agent of several economically important plant diseases, including Pierce's disease (PD) and citrus variegated chlorosis (CVC). Until recently, the inability to transform or produce transposon mutants of X. fastidosa had been a major impediment to identifying X. fastidosa genes that mediate pathogen and plant interactions. A random transposon (Tn5) library of X. fastidosa was constructed and screened for mutants showing more severe symptoms and earlier grapevine death (hypervirulence) than did vines infected with the wild type. Seven hypervirulent mutants identified in this screen moved faster and reached higher populations than the wild type in grapevines. These results suggest that X. fastidosa attenuates its virulence in planta and that movement is important in X. fastidosa virulence. The mutated genes were sequenced and none had been described previously as antivirulence genes, although six of them showed similarity with genes of known functions in other organisms. One transposon insertion inactivated a hemagglutinin adhesin gene (PD2118), which we named HxfA. Another mutant in a second putative X. fastidosa hemagglutinin gene, PD1792 (HxfB), was constructed, and further characterization of these hxf mutants suggests that X. fastidosa hemagglutinins mediate contact between X. fastidosa cells, which results in colony formation and biofilm maturation within the xylem vessels.

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