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  • 1
    Electronic Resource
    Electronic Resource
    Site-specific homologous recombination mutagenesis in group B streptococci (1998)
    Springer
    Methods in cell science 20 (1998), S. 13-20 
    Details
    ISSN: 1573-0603
    Keywords: Capsule synthesis ; Mutagenesis ; Recombination ; Streptococci ; Suicide vector
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We describe the use of suicide vectors to generate site-specific mutations in group B streptococcus. This is accomplished by cloning gene-specific sequences into a temperature sensitive plasmid and selecting for clones which have undergone homologous recombination. The recombinan clones can be easily isolated by selecting for clones which have retained the antibiotic resistance markers that are present on the vector or cloned into the gene-specific sequences. To confirm the fidelity of the recombination events, PCR analysis is performed on chromosomal DNA isolated from the recombinant clones. Using this strategy, we have generated site- specific insertions in several capsule genes and have found that these insertions occurred as expected and that the mutations result in loss of capsule expression. In this report, we specifically detail the construction of cpsB mutants by single and double cross-over recombination and demonstrate that the resulting strains are acapsular.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009810002276
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  • 2
    Electronic Resource
    Electronic Resource
    A simple microtiter plate screening assay for bacterial invasion or adherence (1998)
    Springer
    Methods in cell science 20 (1998), S. 107-111 
    Details
    ISSN: 1573-0603
    Keywords: Bacterial adherence ; Bacterial invasion ; Bacteriological techniques ; Invasion assay ; Microbial colony count ; Soft agar
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Many bacteriologic studies, including cellular invasion and adherence assays, require enumeration of viable organisms or colony forming units. When attempting to screen large numbers of clinical or environmental isolates or laboratory-derived mutants for differences in invasion or adherence phenotype, standard plating methods can be cumbersome and severely limit the number of organisms or conditions which can be tested. As a potential alternative, we describe a simple, rapid and inexpensive soft agar-based technique for semi-quantitative determination of bacterial colony counts directly within the wells of a 96-well microtiter plate.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009830606819
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  • 3
    Electronic Resource
    Electronic Resource
    End-probing: A non-radioactive approach to mapping transposon insertions (1998)
    Springer
    Methods in cell science 20 (1998), S. 113-118 
    Details
    ISSN: 1573-0603
    Keywords: End-labeling ; GBS ; Mapping ; Restriction enzyme mapping ; Tn917
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two pieces of data are needed to fully map the location of a transposon inserted in a plasmid, the site of insertion and the transposon's orientation. Both of these parameters can be determined from a map of restriction sites, which can be derived by end-probing. Like restriction mapping, end-probing reveals the distance between restriction sites on a plasmid. In contrast to restriction mapping, end-probing unambiguously reveals the order of those restriction sites. End-probing is similar to end-labeling, except that the uncertainties inherent in the radiolabeling reaction (and the problems of working with radionucleotides) are avoided. In this paper we discuss the use of end-probing as a means to map insertions of transposon Tn917 into the Streptococcus agalactiae plasmid pGB354
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009851211362
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  • 4
    Electronic Resource
    Electronic Resource
    In vitro systems for investigating group B streptococcal: host cell and extracellular matrix interactions (1998)
    Springer
    Methods in cell science 20 (1998), S. 191-201 
    Details
    ISSN: 1573-0603
    Keywords: Adherence ; Chorioamnion ; Extracellular matrix ; Group B. streptococci ; Invasion fibronectin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Streptococcus agalactiae or group B streptococci (GBS) are gram-positive diplococci and are the leading bacterial cause of pneumoniae, sepsis, and meningitis in neonates. Neonatal GBS infections may occur prior to or during birth. GBS have been cultured from the chorioamnionic membrane of pregnant women and have therefore been associated with chorioamnionitis and premature labor. A potential route for GBS to establish infection of a neonate would be to penetrate the placental membrane of colonized pregnant women. In our laboratory, we have constructed in vitro systems to emulate certain events during the colonization and invasion of host epithelial cell tissues by GBS. By utilizing techniques to grow primary cultures of both chorion cells and amnion cells isolated from human C-section placentas, we have established a relevant model to investigate certain aspects of GBS adherence and invasion into the placental membrane. To identify relevant molecules required for GBS to colonize the multiple tissues it encounters during an infection, we have applied a variety of biochemical approaches with host cell membrane preparations as well as purified extracellular matrix proteins. These techniques are enabling us to further characterize the pathogenic mechanisms utilized by GBS.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009866222748
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