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  • 1
    Keywords: Microbial genetics ; Microbial genomics ; Bacteriology ; Microbial Genetics and Genomics ; Bacteriology ; Springer eBooks
    Description / Table of Contents: Plasmid DNA Isolation and Visualization: Isolation and Characterization of Plasmids from Clinical Samples -- Isolation and Visualization of Plasmids from Gram-Positive Bacteria of Interest in Public Health -- Detection, Isolation, and Characterization of Plasmids in the Environment -- Bacteriophage Isolation and Characterization: Phages of Escherichia coli -- Detection and Characterization of Transposons in Bacteria -- Measuring Plasmid Conjugation Using Antibiotic Selection -- Measuring Plasmid Conjugation Using Fluorescent Reporters -- Methods to Quantify DNA Transfer in Enterococcus -- Quantifying and Characterizing Distributive Conjugal Transfer in Mycobacterium smegmatis -- Spectrophotometric Assays to Quantify the Activity of T4SS ATPases -- Identification of Relaxase-DNA Covalent Complexes and DNA Strand Transfer Reaction Products by Polyacrylamide Gel Electrophoresis -- First Biochemical Steps on Bacterial Transposition Pathways -- Natural Transformation in Escherichia coli -- Integron Identification in Bacterial Genomes and Cassette Recombination Assays -- Methods to Identify and Analyze Vesicle-Protected DNA Transfer -- Measuring Plasmid Stability in Gram-Negative Bacteria -- Methods for the Analysis and Characterization of Defence Mechanisms against Horizontal Gene Transfer: CRISPR Systems -- The Mobilome: Metagenomic Analysis of Circular Plasmids, Viruses, and Other Extrachromosomal Elements -- Identifying Conjugative Plasmids and Integrative Conjugative Elements with CONJscan -- PlasmidFinder and In Silico pMLST: Identification and Typing of Plasmid Replicons in Whole Genome Sequencing (WGS) -- MOBscan: Automated Annotation of MOB Relaxases -- Plasmid Typing and Classification -- Plasmid Reconstruction from Next-Gen Data: A Detailed Protocol for the Use of PLACNETw for the Reconstruction of Plasmids from WGS Datasets -- Statistical Analysis of Accessory Genome -- Inferring Horizontal Gene Transfer with DarkHorse, Phylomizer, and ETE Toolkits -- Methods to Study Fitness and Compensatory Adaptation in Plasmid-Carrying Bacteria -- A Broad Host Range Plasmid-Based Roadmap for ssDNA-Based Recombineering in Gram-Negative Bacteria -- Conjugative Assembly Genome Engineering (CAGE)
    Abstract: This book focuses on technologies used to study horizontal gene transfer (HGT) in prokaryotes. Beginning with a section on the detection and isolation of mobile genetic elements (MGEs), the volume continues with sections concentrating on the analysis of conjugation, transformation, and transduction in HGT as well as a series of methods to analyze the adaptation and evolution of MGEs, with special attention paid to bioinformatics tools. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Horizontal Gene Transfer: Methods and Protocols serves as an ideal guide to the further study of this pervasive, all-important mechanism of genetic originality
    Pages: XV, 413 p. 62 illus., 52 illus. in color. : online resource.
    Edition: 1st ed. 2020.
    ISBN: 9781493998777
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  • 2
    ISSN: 1072-8368
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Relaxases are DNA strand transferases that catalyze the initial and final stages of DNA processing during conjugative cell-to-cell DNA transfer. Upon binding to the origin of transfer (oriT) DNA, relaxase TrwC melts the double helix. The three-dimensional structure of the relaxase domain of TrwC in ...
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  • 3
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] The transfer of DNA across membranes and between cells is a central biological process; however, its molecular mechanism remains unknown. In prokaryotes, trans-membrane passage by bacterial conjugation, is the main route for horizontal gene transfer. It is the means for rapid acquisition of new ...
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Type IV secretion systems (T4SS) are multicomponent transporters of Gram-negative bacteria adapted to functions as diverse as DNA transfer in bacterial conjugation or the delivery of effector proteins into eukaryotic target cells in pathogenesis. The generally modest sequence conservation between T4SS may reflect their evolutionary distance and/or functional divergence. Here, we show that the establishment of intraerythrocytic parasitism by Bartonella tribocorum requires a putative T4SS, which shares an unprecedented level of sequence identity with the Trw conjugation machinery of the broad-host-range antibiotic resistance plasmid R388 (up to 80% amino acid identity for individual T4SS components). The highly conserved T4SS loci are collinear except for the presence of numerous tandem gene duplications in B. tribocorum, which mostly encode variant forms of presumed surface-exposed pilus subunits. Conservation is not only structural, but also functional: R388 mutated in either trwD or trwH encoding essential T4SS components could be trans-complemented for conjugation by the homologues of the B. tribocorum system. Conservation also includes the transcription regulatory circuit: both T4SS loci encode a highly homologous and interchangeable KorA/KorB repressor system that negatively regulates the expression of all T4SS components. This striking example of adaptive evolution reveals the capacity of T4SS to assume dedicated functions in either DNA transfer or pathogenesis over rather short evolutionary distance and implies a novel role for the conjugation systems of widespread broad-host-range plasmids in the evolution of bacterial pathogens.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 39 (2001), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The mobilization region of plasmid CloDF13 was localized to a 3.6 kb DNA segment that was analysed by transposon mutagenesis and DNA sequencing. Analysis of the DNA sequence allowed us to identify two mobilization genes and the CloDF13 origin of conjugative transfer (oriT), which was localized to a 661 bp segment at one end of the mobilization (Mob) region. Thus, the overall organization was oriT–mobB–mobC. Plasmid CloDF13 DNA was isolated mainly as a relaxed form that contained a unique strand and site-specific cleavage site (nic). The position of nic was mapped to the sequence 5′-GGGTG/GTCGGG-3′ by primer extension and sequencing reactions. Analysis of Mob− insertion mutants showed that mobC was essential for CloDF13 relaxation in vivo. The sequence of mobC predicts a protein (MobC) of 243 amino acids without significant similarity to previously reported relaxases. In addition to MobC, the product of mobB was also required for CloDF13 mobilization and for oriT relaxation in vivo. mobB codes for a protein (MobB) of 653 amino acids with three predicted transmembrane segments at the N-terminus and the NTP-binding motifs characteristic of the TraG family of conjugative coupling proteins. Membership of the TraG family was confirmed by the fact that CloDF13 mobilization by plasmid R388 was independent of TrwB and only required PILW. However, contrary to the activities found for other coupling proteins, MobB was required for efficient oriT cleavage in vivo, suggesting an additional role for this particular protein during oriT processing for mobilization. Additionally, the cleavage site produced by the joint activities of MobB and MobC was shown to contain unblocked ends, suggesting that no stable covalent intermediates between relaxase and DNA were formed during the nic cleavage reaction. This is the first report of a conjugative transfer system in which niccleavage results in a free nicked-DNA intermediate.
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  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: IS91 displays a number of characteristics unique among insertion sequence (IS) elements, suggesting that it transposes by a novel mechanism called rolling-circle (RC) transposition. We reported previously that IS91 transposase (TnpA) amino acid sequence shares a series of five conserved signatures with A proteins of RC replicating phages, including a pair of invariant tyrosines that catalyse two successive transesterification reactions during replication initiation and termination. To analyse their role in IS91 transposition, we constructed a series of TnpA derivatives in which the invariant Tyr-249 and/or Tyr-253 were mutated to either phenylalanine or serine. Mutation of either tyrosine resulted in complete loss of transposition activity in vivo. This result was taken as a first new line of evidence that TnpA is a functional analogue of φX174 phage A protein. Secondly, RC replication plasmids and phages accumulate single-stranded DNA (ssDNA) intermediates as a result of uncoupled leading and lagging DNA strand synthesis. Using a plasmid carrying an IS91-derived IRLkan-IRR transposable cassette, in which the left (IRL)- and right (IRR)-terminal sequences of IS91 flank a kanamycin resistance gene (kan), we demonstrated the in vivo formation of two new DNA species after induction of transposase expression. The first was a circular ssDNA that contained the transposable cassette covalently joined at its exact termini, whereas the second was a double-stranded circle of the same element. When this experiment was repeated using the mutant transposases described above, the ssDNA and dsDNA intermediates could not be observed, indicating that the integrity of both Y249 and Y253 was essential for their appearance. The presence of ssDNA intermediate products is the first biochemical evidence for a RC mechanism of IS91 transposition.
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  • 7
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A series of plasmids carrying an IRL-kan-IRR transposable cassette, in which IRL and IRR are the left- and right-terminal sequences of IS91, have been constructed. These cassettes could be complemented for transposition with similar efficiency when IS91 transposase was provided either in cis or in trans. A total of 87% of IS91 transposition products were simple insertions of the element, while the remaining 13% were plasmid fusions and co-integrates. When transposase expression was induced from an upstream lac promoter, transposition frequency increased approximately 100-fold. An open reading frame (ORF) present upstream of the transposase gene, ORF121, could be involved in target selection, as mutations affecting this ORF were altered in their insertion specificity. Intramolecular rearrangements were analysed by looking at transposition events disrupting a chloramphenicol resistance gene (cat ) located outside the transposable cassette. Plasmid instability resulting from insertion of an extra copy of IRL-kan-IRR within the cat gene was observed; transposition products contained a second copy of the cassette inserted either as a direct or as an inverted repeat. No deletion or inversion of the intervening DNA was observed. These results could be explained as a consequence of intramolecular transposition of IS91 according to a model of rolling-circle transposition.
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford UK : Blackwell Science Ltd
    Molecular microbiology 24 (1997), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The conjugative transfer region of the IncX plasmid R6K (TRAX) was analysed by transposon mutagenesis and DNA sequencing. Tn5tac1 insertional mutations localized TRAX to a 14.8 kb segment containing the α origin of transfer (oriTα), genes involved in conjugative DNA-processing (DtrX) and genes involved in pilus synthesis and assembly (MpfX). A second functional oriT, oriTβ, was located at a distance of 5.3 kb from oriTα and was outside TRAX. MpfX occupied a segment of 10 kb, as judged by the location of insertions conferring resistance to infection by the X pilus-specific phage X-2. At both sides of MpfX there were insertions that were Tra− but X-2 sensitive, suggesting that the mutations were in DtrX. This region was sequenced and three genes were identified: taxA, taxB, and taxC. The overall organization was oriTα–taxA–taxC–MpfX–taxB. taxC coded for a oriT-relaxase that belongs to the VirD2 family. taxA coded for a protein of 181 amino acids that showed similarity to TraY of F-like plasmids and to the Arc-repressor superfamily. TaxB showed similarity to TraG-like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA-transport apparatus. TaxA and TaxC are required for oriT nicking in vivo. The nicking reaction was mistakenly assumed by Flashner et al. (1996) to represent a feature of the vegetative replication origins. However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K. Conversely, protein π, which is absolutely required for replication of R6K, was not required for conjugative transfer. In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation. Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein π for conjugation.
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  • 9
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: MbeA is a 60 kDa protein encoded by plasmid ColE1. It plays a key role in conjugative mobilization. MbeA*, a slightly truncated version of MbeA, was purified for in vitro analysis. MbeA* catalysed DNA cleavage and strand-transfer reactions using oligonucleotides embracing the ColE1 nic site, which was mapped to 5′-(1469)CTGG/CTTA(1462)-3′. Thus MbeA is the relaxase for ColE1 conjugal mobilization, in spite of the fact that it lacks a three histidine motif considered the invariant signature of conjugative relaxases. Amino acid sequence comparisons suggest MbeA is nevertheless related to the common relaxase protein family. For instance, MbeA residue Y19 could correspond to the invariant tyrosine in Motif I, whereas H97, E104 and N106 may constitute the equivalent residues to the histidine triad in Motif III. This hypothesis was tested by site-directed mutagenesis. MbeA amino acid residues Y19, H97, E104 and N106 were changed to alanine. MbeA mutant N106A showed reduced oligonucleotide cleavage and strand-transfer activities, whereas mutation in the other three residues resulted in proteins without detectable activity, suggesting they  are  directly  implicated  in  catalysis  of  DNA-cleavage and strand-transfer reactions. A double substitution of E104 and N106 by histidines, therefore reconstituting the canonical histidine triad, restored relaxase activities to 1% of wild type. Thus, MbeA is a    variant    of    the    common    relaxase    theme    with    a HEN signature motif, which has to be added to the canonical three histidine motif of previously reported relaxases.
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  • 10
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Relaxosomes are specific nucleoprotein structures involved in DNA-processing reactions during bacterial conjugation. In this work, we present evidence indicating that plasmid R388 relaxosomes are composed of origin of transfer (oriT) DNA plus three proteins TrwC relaxase, TrwA nic-cleavage accessory protein and integration host factor (IHF), which acts as a regulatory protein. Protein IHF bound to two sites (ihfA and ihfB) in R388 oriT, as shown by gel retardation and DNase I footprinting analysis. IHF binding in vitro was found to inhibit nic-cleavage, but not TrwC binding to supercoiled DNA. However, no differences in the frequency of R388 conjugation were found between IHF− and IHF+ donor strains. In contrast, examination of plasmid DNA obtained from IHF− strains revealed that R388 was obtained mostly in relaxed form from these strains, whereas it was mostly supercoiled in IHF+ strains. Thus, IHF could have an inhibitory role in the nic-cleavage reaction in vivo. It can be speculated that triggering of conjugative DNA processing during R388 conjugation can be mediated by IHF release from oriT.
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