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  • Blackwell Publishing Ltd  (6)
  • 1990-1994  (6)
Document type
Publisher
Years
Year
  • 1
    Electronic Resource
    Electronic Resource
    Transformation-mediated exchange of virulence determinants by co-cultivation of pathogenic Neisseriae (1992)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 100 (1992), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract The horizontal flow of genetic material between microbes utilizes three principal routes: conjugation, transduction and transformation. While the significance in nature of the first two pathways is generally accepted, the in vivo role of transformation remains uncertain, despite the early observations by Griffith in 1928 on the transformation of streptococci from an avirulent to a virulent state [1]. Recently, circumstantial evidence was collected suggesting a role for transformation-mediated horizontal exchange in the modulation of virulence determinants of pathogenic Neisseriae and the variation of surface structures. In order to further assess the significance of transformation-mediated exchange we performed simple co-cultivation experiments of different Neisseria strains. We observed an efficient intra- and interspecies transfer of essential virulence determinants; the process was sensitive to the presence of DNaseI in the culture and was blocked in transformation-deficient recipients.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.1992.tb14062.x
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  • 2
    Electronic Resource
    Electronic Resource
    Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 8 (1993), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Three out of 10 Helicobacter pylori clinical isolates were found to be naturally competent for genetic transformation to streptomycin resistance by chromosomal DNA extracted from a spontaneous streptomycin-resistant H. pylori mutant. The frequency of transformation varied between 5 × 10−4 and 4 × 10−6, depending on the H. pylori isolate used. Transposon shuttle mutagenesis based on this natural competence was established using the flagellin gene flaA as the target. The cloned flaA gene was interrupted by insertion of TnMax1, a mini-Tn1721 transposon carrying a modified chloramphenicol-acetyltransferase gene, the catGC cassette. Natural transformation of competent H. pylori strains with plasmid constructs harbouring a catGC-inactivated flaA gene resulted in chloramphenicol-resistant transformants at an average frequency of 4 × 10−5. Southern hybridization experiments confirmed the replacement of the chromosomal H. pylori flaA gene by the cat-inactivated cloned gene copy via homologous recombination resulting in allelic exchange. Phenotypic characterization of the mutants demonstrated the absence of flagella under the electron microscope and the loss of bacterial motility. Immunoblots of cell lysates of the H. pylori mutants with an antiserum raised against the C-terminal portion of recombinant H. pylori major flagellin (FlaA) confirmed the absence of the 54kDa FlaA protein. This efficient transposon shuttle mutagenesis procedure for H. pylori based on natural competence opens up new possibilities for the genetic assessment of putative H. pylori virulence determinants.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1993.tb01618.x
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    Paper (German National Licenses)
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  • 3
    Electronic Resource
    Electronic Resource
    Interaction of two variable proteins (PilE and PilC) required for pilus-mediated adherence of Neisseria gonorrhoeae to human epithelial cells (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: Pili confer the initial ability of Neisseria gonorrhoeae to bind to epithelial cells. Pilin (PilE), the major pilus subunit, and a minor protein termed PilC, reportedly essential for pilus biogenesis, undergo intra-strain phase and structural variation. We demonstrate here that at least two different adherence properties are associated with the gonococcal pili: one is specific for erythrocytes, which is virtually unaffected by PilE variation, and another is specific for epithelial cells, and is modulated in response to the variation of PilE. Based on this finding, mutants of a recA - strain were selected that had lost the ability to bind to human cornea epithelial cells (A-) but retained the ability to form pili (P+) and to agglutinate human erythrocytes (H+). The adherence-negative mutants failed to produce detectable levels of PMC1 or PilC2 proteins, representing pilC phase variants generated in the absence of RecA. The A-pilC phase variants were indistinguishable from their A+parents and spontaneous A+ revertants with regard to the amount of PilE produced and its electrophoretic mobility, the degrees of piliation and haemagglutination, and the pilE nucleotide sequence. These data demonstrate a central role for PilC in pilus-mediated adherence of N. gonorrhoeae to human epithelial cells and further indicate that neither PNC1 nor PilC2 is obligatory for the assembly of gonococcal pili.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1992.tb02211.x
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  • 4
    Electronic Resource
    Electronic Resource
    Novel determinant (comA) essential for natural transformation competence in Neisseria gonorrhoeae and the effect of a comA defect on pilin variation (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 10 (1993), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A novel genetic determinant (comA) has been identified and found to be required for the transformation of piliated Neisseria gonorrhoeae. Mutants in comA of strain MS11 grow normally and are DNA-uptake proficient but blocked in the translocation of DNA into the cytoplasm. Here we show by site-specific mutagenesis and genetic complementation that only one of two open reading frames identified in comA is essential for competence: it encodes a protein (ComA) with a predicted size of 74kDa. The comAgene maps upstream of the iga locus and is transcribed in the opposite orientation, probably under the control of a putative σ;54 type promoter. While DNA probes specific for the N. gonorrhoeae iga locus reveal only a little cross-reactivity with commensal Neisseria species, the neighbouring comA gene appears to be present in most of them. ComA fusion proteins were obtained by in vitro translation. The synthesized gene products migrated atypically in SDS gels indicating its strong hydrophobicity. Several transmembrane α-helices were predicted from the amino acid sequence of ComA which, in the context of an observed sequence similarity with other inner membrane proteins, suggests a location for the protein in the inner membrane. Using piliated and non-piliated comA mutants the consequences of transformation deficiency on pilin phase variation were assessed. We show that the comA defect affects some but not all types of DNA rearrangements associated with pilE variation. The results are in agreement with previous observations supporting the notion that multiple recombination pathways contribute to the variability of pilE.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1993.tb00942.x
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  • 5
    Electronic Resource
    Electronic Resource
    Immunogenicity and evolutionary variability of epitopes within IgA1 protease from serogroup A Neisseria meningitidis (1994)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 11 (1994), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Five murine epitopes were defined and mapped within IgA1 protease produced by Neisseria meningitidis. Epitopes 1 and 2 were present in IgA1 protease from all strains, and from Neisseria gonorrhoeae. Epitopes 3 through to 5 varied between subgroups of serogroup A meningococci. but have remained constant over decades within the subgroups, except for epitope 4, which changed between 1983 and 1987 during the spread of subgroup III meningococci from Asia to Africa. Binding of monoclonal antibodies to epitopes 1, 4 and 5 neutralized enzymatic function. Human sera containing antibodies to lgA1 protease as a result of natural infection inhibited binding of monoclonal antibodies to epitope 4 but not to the other epitopes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1994.tb00299.x
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  • 6
    Electronic Resource
    Electronic Resource
    Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants (1994)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 12 (1994), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: One requirement for the invasion of, and tight adherence to, human epitheiial cells by Neisseria gonorrhoeae is the synthesis of distinct opacity (Opa) outer membrane proteins, encoded by a family of phase-variable chromosomal genes. However, cloning and surface expression of invasion-promoting Opas in Escherichia coli is not sufficient for the efficient invasion of epithelial cells: additional factors besides Opa may be involved in this process. Using the phoA mini-transposon TnMax4, a library of gonococcal mutants affected in the expression of genes encoding exported proteins was generated through shuttle mutagenesis. Of a total of 608 PhoA+ plasmid clones identified in E. coli E145 approximately 40% were used successfully in transforming N. gonorrhoeae and in activating the corresponding chromosomal genes. Gonococci producing the invasion-promoting Opa50 served as the genetic background to identify 51 mutants unable to enter Chang human epithelial cells. We expect some of these mutations affect the interaction of N. gonorrhoeae with epithelial cells directly, while other mutants may carry defects in general house-keeping, secretory and/or regulatory determinants. In some mutants the loss of invasiveness appears to be due to a negative dominant effect of the PhoA+ fusions produced in these mutants. Some of the identified genes display a phase-variation phenomenon in E. coli and several genes are found in multiple copies in N. gonorrhoeae and/or present only in pathogenic Neisseria species.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1994.tb01068.x
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