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  • American Institute of Physics  (70)
  • Blackwell Publishing Ltd  (14)
  • American Association for the Advancement of Science (AAAS)  (2)
  • EMBO Press  (1)
  • 1
    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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  • 2
    Electronic Resource
    Electronic Resource
    Cloning of a surface protein antigen gene from serotype c Streptococcus mutans (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The structural gene for a 190kD protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was cloned into the plasmid vector pUC118. SDS-polyacrylamide gel electrophoresis and Western immunoblotting showed that the Escherichia coli harbouring the chimaeric plasmid produced multiple polypeptides of 190-210kD. Immunodiffusion analysis revealed that the cloned PAc had the same specific determinants as S. mutans PAc. The cloned pac gene was mapped, and its transcriptional orientation was determined by characterizing deletion mutants of the chimaeric plasmid. Southern blot analysis with the cloned gene sequence as a probe revealed the presence of a homologous sequence in DNAs from sero-types e and f S. mutans. PAc-defective mutants were constructed by inserting an erythromycin-resistance gene into the pac gene. The cell-surface hydrophobicity of the mutants was lower than that of the parent strain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1989.tb01811.x
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  • 3
    Electronic Resource
    Electronic Resource
    Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis (1993)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 9 (1993), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Bacillus anthracis produces a gamma-linked poly-D-glutamic acid capsule that is essential for virulence. A 6.2 kb fragment of B. anthracis DNA (cap), when present in Escherichia coli, produces a capsular polymer that is immunologically identical to that produced by B. anthracis. By immunodiffusion analysis of E. coli strains carrying varying portions of the cap region, we identified a novel gene (dep) responsible for degradation of the capsular polymer of B. anthracis. The simultaneous presence of the cap region and the dep gene caused production of low-molecular-weight, degraded capsular polymer both in E. coli and in B. anthracis, whereas the cap region atone caused production of a high-molecular-weight capsule. The dep gene mapped immediately downstream of the cap region within a 1.8 kb fragment and was transcribed in the same direction. This fragment was sequenced and a 1401 bp open reading frame (ORF) was found that is predicted to encode a peptide with molecular weight of 51460. By in vitro transcription-translation analysis, this ORF was shown to be the dep gene product. The deduced amino acid sequence of the dep product has sequence similarity to E. coli and mammalian γ-glutamyltranspeptidase (GGT). However, the Dep protein did not have GGT activity. The Dep protein appears to be an enzyme that catalyses the hydrolysis of the poly-D-glutamic acid capsule. Although the biological functions of the dep gene are unknown, it is possible that low-molecular-weight, diffusible polyglutamates produced through the action of the dep gene may act to inhibit host defence mechanisms.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1993.tb01710.x
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  • 4
    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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  • 5
    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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  • 6
    Electronic Resource
    Electronic Resource
    A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The expression of plasmid-encoded, invasion-related antigens lpa b, c and d of Shigella flexneri was found to be positively regulated at transcriptional level by a 33kD protein produced by the previously defined, virulence-associated Region 1 on the SalI fragment B of the 230 kb invasion plasmid. The gene (designated virB) was identified and its nucleotide sequence determined. No Ipa b or c was produced in the absence of an intact virB gene although lower levels of d were produced. The previously reported regulatory activity of the virF gene some 30 kb distance away was shown to act exclusively through virB. In contrast, the activation of the virG gene necessary for intercellular spread occurred directly by virF without the requirement for virB. This study thus ascribes a critical function to a previously recognized, but functionally undefined, virulence locus on the large invasion plasmid of S. flexneri. The virF gene appears to have a central role in activation of the 230kb plasmid-encoded virulence genes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1989.tb00210.x
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  • 7
    Electronic Resource
    Electronic Resource
    Molecular characterization of a surface protein antigen gene from serotype c Streptococcus mutans, implicated in dental caries (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The complete nucleotide sequence of the gene for a cell-surface protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was determined. The pac gene consisted of 4695 bp and coded for a 170 773 D protein. The pac gene product contained a putative 38 amino acid signal peptide, resulting in a 166817D mature protein. A potential promoter sequence and a putative Shine-Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in the PAc. One repeating region located in the N-terminal region was rich in alanine, and the other located in the central region was rich in proline. Southern blot analysis under the less stringent condition (allowing up to 35% base mismatch) revealed that the probe covering the prolinerich region hybridized to DNA preparations from strains of Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei as well as Streptococcus mutans.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1989.tb00215.x
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  • 8
    Electronic Resource
    Electronic Resource
    Cloning and CO2-dependent expression of the genetic region for encapsulation from Bacillus anthracis (1988)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 2 (1988), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The capsule of Bacillus anthracis is an important virulence factor consisting of poly-o-glutamic acid. The genetic region required for the encapsulation was cloned in Escherichia coli from the capsule plasmid pTE702, using a selection procedure based on an immunodlffusion assay. The cloned region directed synthesis of the capsule both In E. coli and B. anthracis. Capsule synthesis from these clones, as in the wild type, was dependent upon the presence of CO2. However, encapsulation directed by the cloned fragment was less marked than from pTE702. Another region enhancing capsulation was shown to exist on pTE702. The minimum size of the encapsulation region was defined to within 2.7 kb DNA and shown to be essential for the encapsulation in B. anthracis.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1988.tb00041.x
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  • 9
    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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  • 10
    Electronic Resource
    Electronic Resource
    Functional organization and nucleotide sequence of virulence Region-2 on the large virulence plasmid in Shigella flexneri 2a (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 3 (1989), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The 7kb virulence Region-2 of the large (virulence) plasmid in Shigella flexneri 2a encodes several proteins required for invasion of intestinal epithelial cells. Insertion and deletion mutagenesis, DNA subcloning and SDS-polyacrylamide gel electro-phoresis of proteins synthesized in minicells demonstrated five genes in this region. They encode 24, 18, 62 (lpaB), 41 (lpaC) and 37 (lpaD)-kiloDalton (kD) proteins. Complementation of Tn5-induced mutations in Region-2 with the above plasmid constructs indicated that Region-2 consists of two operons and that the three lpa proteins are essential for the virulence phenotype. The transcriptional organization determined by Northern blotting, S1 nuclease protection and the effect of Tn5 insertions on expression of the lpa proteins revealed that Region-2 has three promoters that transcribe RNAs of 4.0, 4.5 and 7.5kb. The 4.0 kb RNA was the transcript for the operon encoding the 24, 18 kD, lpaB and C proteins and the 4.5 kb RNA for the ipsD gene. In addition, the full-length RNA of 7.5 kb which covers Region-2 supplemented full expression of the lpa proteins. The 7663 nucleotides of Region-2 were determined to confirm the five open reading frames encoding 23655, 17755, 62168, 41077 and 36660 Dalton proteins, respectively, and their regulatory sequences.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1989.tb00269.x
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