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  • Articles  (5)
  • Blackwell Publishing Ltd  (4)
  • Thomas Telford  (1)
  • 1990-1994  (5)
  • 1
    Electronic Resource
    Electronic Resource
    Identification and characterization of virK, a virulence-associated large plasmid gene essential for intercellular spreading of Shigella flexneri (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 6 (1992), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Seven virulence loci have been identified by Tn5 insertion mutagenesis on the large 230 kb plasmid (pMYSH6000) of Shigella flexneri 2a. In this study, we used Tn10 insertion mutagenesis and identified a novel virulence locus on pMYSH6000 responsible for bacterial spread. Characterization of the invading bacteria of the Tn10 insertion mutants in the epithelial cells revealed that the bacteria were capable of at least some intracellular spreading but not intercellular spreading. Immunoblot analysis of lysates of the Tn10 insertion mutants with a VirG-specific antipeptide antibody revealed diminished levels of the 116 kDa VirG protein. The virG mRNA in the mutants, however, was expressed at the same level as that in the wild type. The DNA region required for the virulence phenotype was localized to a 1.6 kb DNA sequence in the Sal I-K fragment on the plasmid, and thus the locus was designated virK. Expression of virK in Escherichia coli using a T7 RNA polymerase-dependent promoter system yielded a 36 kDa protein. The nucleotide sequence of 1642 bp encoding VirK function was determined, and an open reading frame encoding 316 amino acid residues was shown to encode the VirK protein. The virK region was highly conserved among the large virulence plasmids of shigellae and enteroinvasive Escherichia coli. These results suggest that VirK function is an essential virulence determinant for shigellae Involved in the expression of virG gene product at post-transcriptional level.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1992.tb01413.x
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Construction of a physical map of the chromosome of Shigella flexneri 2a and the direct assignment of nine virulence-associated loci identified by Tn5 insertions (1991)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 5 (1991), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: To establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a Notl restriction map of the chromosome was constructed by exploiting Notl-linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K-12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K-12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci Identified previously by random Tn5 insertions were physically mapped on the chromosome. To demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K-12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5′ and 3’flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison of genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K-12.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1991.tb02147.x
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  • 3
    Electronic Resource
    Electronic Resource
    Virulence-associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis (1991)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 5 (1991), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Shigellae are the causative agents of bacillary dysentery and are capable of invading epithelial cells, multiplying therein and spreading into adjacent cells. To identify genes on the chromosome associated with the virulence phenotype, 9114 independent Tn5 insertion mutants were isolated in a virulent strain of Shigella flexneri. By using an in vitro assay for intercellular spread or an animal infection model, the Serény test, 50 chromosomal Tn5 mutants with reduced virulence were identified. The 50 mutants were characterized with respect to their virulence phenotypes, including three different mutations that affect invasion of epithelial cells, bacterial metabolism and structure of lipopolysaccharide. Mutants with reduced invasive ability were further characterized and it was found that two of them had decreased levels of IpaB, C and D antigens as well as the mRNA for the ipaBCD operon encoded by the large virulence plasmid, suggesting that positive regulatory elements for the IpaBCD operon are encoded by the chromosome. Assignment of the 50 Tn5 insertions of the mutants to the 19 Notl restriction fragments of the chromosomal DNA has permitted the identification of at least nine virulence-associated chromosomal loci.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1991.tb01839.x
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  • 4
    Electronic Resource
    Electronic Resource
    Temperature-regulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large plasmid (1991)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 5 (1991), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The invasion phenotype of shigellae is subject to thermoregulation that is known to be expressed through activation of some invasion (inv) genes such as ipaB, ipaC, and ipaD encoded by the large virulence plasmid of Shigeila flexneri. The expression of ipa genes is regulated positively by virF through the activation of virB on the plasmid. To identify the mediator for the thermoregulation of the large plasmid, we have studied the effect of temperature on the transcription of virF and virB genes and ipa and the other two inv operons. The results showed that transcription of VirB was affected by temperature more strictly than that of virF. Analysis of the mRNA level of virB at different levels of virF transcription indicated that virB transcription depended upon both temperature and virF. On the other hand, transcriptions of ipa and the other two inv operons depended on the activation of virB transcription but not on temperature. By inducing wr8 transcription from a tac promoter fused with the virB region, invasion ability was restored to a WrF-deletion mutant at 30°C as well as at 37°C. By using conditions in which the temperature-dependent expression of the invasion phenotype was circumvented by the induction of virB transcription, intercellular spreading ability in a virF+, virB::Tn5 strain was shown to be expressed even at 30°C. These results suggest that the virB transcription stage is the main target for the thermoregulation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1991.tb00762.x
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  • 5
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    Unknown
    Energy dissipation in inelastic flow of saturated cohesionless granular media (1994)
    Thomas Telford
    Details
    Publication Date: 1994-03-01
    Print ISSN: 0016-8505
    Electronic ISSN: 1751-7656
    Topics: Architecture, Civil Engineering, Surveying , Geosciences
    Published by Thomas Telford
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