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  • 1990-1994  (512,735)
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Years
Year
  • 1
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Physics Letters B 294 (1992), S. 466-478 
    ISSN: 0370-2693
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Physics Letters B 317 (1993), S. 474-484 
    ISSN: 0370-2693
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 3
    Call number: 01-Beschaffungsw:70 ; M230:3/1 ; M230:3/2
    Pages: loose-leaf
    ISBN: 3-8073-0843-1
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    01-Beschaffungsw:70 departmental collection or stack – please contact the library
    M230:3/1 departmental collection or stack – please contact the library
    M230:3/2 departmental collection or stack – please contact the library
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  • 4
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    Totowa, NJ : Humana Press
    Keywords: Molecular Biology / methods
    Notes: This is a series title, single volumes see link below.
    ISSN: 1940-6029
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  • 5
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    New York, NY : Elsevier
    Keywords: Biochemistry ; Enzymes
    Notes: This is a series title, single volumes see link below.
    ISSN: 1557-7988
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  • 6
    Keywords: Leukemia / therapy ; Prognosis
    Notes: Last volumes with varying subtitle and editor.
    ISSN: 0949-7021
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  • 7
    Keywords: Hazardous Substances / toxicity
    Notes: Ceased with vol. 15(1999).
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  • 8
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    Königswinter : Petersberg Verlag
    Keywords: Arbeitsgemeinschaft der Großforschungseinrichtungen (Germany) ; Research institutes / Germany ; Research ; Germany
    Notes: Ceased with ed. 1995.
    ISSN: 0935-2236
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 10 (1994), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: Exploiting an incremental and parallel processing scheme is useful to improve the performance of natural language generation systems. TAG-GEN is a TAG-based syntactic generator that realizes both principles. It is shown how the demands of incremental and parallel generation influence the definition, the design, and the processing of syntactic rules on the basis of tree-adjoining grammars.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 10 (1994), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: Natural-language generation has gained importance since AI systems have become more sophisticated and canned text can no longer serve the elaborate communicative tasks in such a system. In this paper, suggestions are presented to apply uniformly the formalism of tree-adjoining grammar (TAG) for all tasks of natural-language generation. Up to now plan-based systems have been preferred for most of the processing during generation. Since the individual tasks of natural-language generation are very different and complex; for example, the determination of the propositional structure of a text is described using the TAG formalism–extended by constraints and feature descriptions (UCTAC). Nevertheless, this paper proposes basic ideas for the generalization of applying a grammar formalism to natural-language generation in order to build an overall integrated system for natural-language generation. Finally, advantages of such a uniform model are discussed.
    Type of Medium: Electronic Resource
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  • 11
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 10 (1994), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 12
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 10 (1994), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: This paper presents an evaluation of a heuristic for partial-order planning, known as temporal coherence. The temporal coherence heuristic was proposed by Drummond and Currie as a method to improve the efficiency of partial-order planning without losing the ability to find a solution (i.e., completeness). It works by using a set of domain constraints to prune away plans that do not “make sense,” or are temporally incoherent. Our analysis shows that, while intuitively appealing, temporal coherence can only be applied to a very specific implementation of a partial-order planner and still maintain completeness. Furthermore, the heuristic does not always improve planning efficiency; in some cases, its application can actually degrade the efficiency of planning dramatically. To understand when the heuristic will work well, we conducted complexity analysis and empirical tests. Our results show that temporal coherence works well when strong domain constraints exist that significantly reduce the search space, when the number of subgoals is small, when the plan size is not too large, and when it is inexpensive to check each domain constraint.
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  • 13
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 10 (1994), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Notes: The frequent and conventional use of nonliteral language has been a major stumbling block for natural language processing systems since the early machine translation efforts. Metaphor, metonymy, and indirect speech acts are among the most troublesome phenomena. Recent computational efforts addressing these problems have taken an approach that emphasizes the use of systematic knowledge about nonliteral language conventions. We are currently engaged in an effort to supply this knowledge in the case of conventional metaphor. We are constructing MetaBank: an empirically derived and theoretically motivated knowledge-base of English metaphorical conventions. This article describes our three-part approach to the construction of MetaBank: the collection of on-line textual resources and databases of linguistic generalizations, the development of a methodology for analyzing these resources, and the construction of a knowledge-base based on the preceding analyses.
    Type of Medium: Electronic Resource
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  • 14
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 10 (1994), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 15
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 10 (1994), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 16
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 10 (1994), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 17
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Computational intelligence 10 (1994), S. 0 
    ISSN: 1467-8640
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Computer Science
    Type of Medium: Electronic Resource
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  • 18
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 16 (1994), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 19
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 16 (1994), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Notes: For many physical processes it is extreme levels which are of greatest concern. This article gives a practical introduction to the problems and models involved.
    Type of Medium: Electronic Resource
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  • 20
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 16 (1994), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 21
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 16 (1994), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 22
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 16 (1994), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 23
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 16 (1994), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Notes: The instructor can help begining students master the numerous results of regression analysis by using helpful notation and by providing a framework for organising regression outputs. For the latter, model comparison is a useful operating notion.
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  • 24
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 16 (1994), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Notes: Several estimates of an unknown population size are compared.
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  • 25
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 16 (1994), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Type of Medium: Electronic Resource
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  • 26
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Teaching statistics 16 (1994), S. 0 
    ISSN: 1467-9639
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Mathematics
    Notes: This work demonstrates common elements correlation, its extension to negative correlation, and the production of bivariate normal samples for a specified correlation matrix.
    Type of Medium: Electronic Resource
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  • 27
    ISSN: 0550-3213
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 28
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Nuclear Physics, Section B 421 (1994), S. 3-37 
    ISSN: 0550-3213
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 29
    Electronic Resource
    Electronic Resource
    Amsterdam : Elsevier
    Nuclear Physics, Section B 418 (1994), S. 403-427 
    ISSN: 0550-3213
    Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
    Topics: Physics
    Type of Medium: Electronic Resource
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  • 30
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In most soft-rotting Erwinia spp., including E. carotovora sub sp. carotovora strain 71 (Ecc71), production of the plant cell wall degrading enzyme pectin lyase (PnI) is activated by DNA-damaging agents such as mitomycin C (MC). Induction of PnI production in Ecc71 requires a functional recA gene and the rdg locus DNA sequencing and RNA analyses revealed that the rdg locus contains two regulatory genes, rdgA and rdgB, in separate transcriptional units. There is high homology between RdgA and repressers of lambdoid phages, specially φ80. RdgB, however, has significant homology with transcriptional activators of Mu phage. Both RdgA and RdgB are also predicted to possess helix-turn-helix motifs. By replacing the rdgB promoter with the IPTG-inducible tac promoter, we have determined that rdgB by itself can activate PnI production in Escherichia coli. However, deletion analysis of rdg+ DNA indicated that, when driven by their native promoters, functions of both rdgA and rdgB are required for the induction of pnIA expression by MC treatment. While rdgB transcription occurs only after MC treatment, a substantial level of rdgA mRNA is detected in the absence of MC treatment. Moreover, upon induction with MC, a new rdgA mRNA species, initiated from a different start site, is produced at a high level. Thus, the two closely linked rdgA and rdgB genes, required for the regulation of PnI production, are expressed differently in Ecc71.
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  • 31
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: RNA secondary structure is important in a wide variety of biological processes, but relatively little is known about the pathways and kinetics of RNA folding. When the IS 10 transposase (tnp) gene is transcribed from a promoter outside the element, little increase in tnp expression is observed. This protection from outside transcription (pot) occurs at the translational level, presumably resulting from mRNA secondary structure proposed to sequester the tnp ribosomebinding site. Here, we confirm the pot RNA structure and show that it blocks 30S ribosomal subunit binding in vitro. Point mutations that abolish protection in vivo map to the pot structure. Surprisingly, these pot mutations do not severely alter the pot secondary structure or increase 30S subunit binding in vitro, except in one case. Using an oligonucleotide hybridization assay, we show that most of the pot mutations slow the kinetics of pot structure formation, with little or no effect on the inhibitory function of the final structure. Moreover, a suppressor mutation reverses this effect. We propose a pathway for pot mRNA folding that is consistent with the mutations and implicates the formation of important kinetic intermediates. The signficance of these observations for the RNA folding problem in general is discussed.
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  • 32
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Transcriptional control of the himA and the himD/hip genes coding for the two subunits of the integration host factor (IHF) was investigated. The promoters for the two genes were identified by the use of primer extension and S1 analysis. Expression from both promoters was found to increase as the cells enter stationary phase. Mutation in rpoS, known to be induced upon entry to stationary phase, dramatically reduced the growth-phase response of the himA P4 promoter but had only a small effect on the induction of the himD/hip promoter. The increased activity of both promoters required the presence of the rel4 and spoT genes, suggesting that ppGpp plays a major role in the response to stationary phase. An artificial increase in ppGpp in exponentially growing cells induced a rapid increase in himA P4 and himD/hip mRNA levels. Experiments with a mutant defective in rpoS showed that the response of the himA P4 promoter to high ppGpp levels was greatly reduced while that of himD/hip was only slightly affected. Therefore, it seems that different mechanisms involving RpoS and ppGpp regulate the growth-phase response of the two promoters. We propose that the effect of ppGpp on himA P4 is mediated via RpoS whereas the himD/hip promoter is affected by ppGpp independently of RpoS.Expression of the himD/hip and himA genes was found to be subject to negative autoregulation. IHF-binding sites, implicated in autoregulation, were found to overlap both the himD/hip and himA P4 promoters. An additional IHF-binding site was found upstream of the himD/hip promoter. AM three sites show low binding affinity to IHF suggesting that autoregulation can take place only after sufficiently high levels of IHF accumulate in the cell.
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  • 33
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Gonococcal porins (Por) from strains FA19 (Por-1, serogroup A), MS11 (Por-2, serogroup B) and FA6434 (Por-5, a hybrid porin containing epitopes from both serogroups), were expressed in Escherichia coli and purified under non-denaturing conditions. Porins were inserted into liposomes, and they were bound by monoclonal antibodies which bind native Por and intact gonococci, but not denatured Por. All three recombinant porins (rPor) were highly immunogenic in rabbits without additional adjuvant. The rPor antisera were specific for Por by Western blotting and whole-cell radioimmunoprecipitation and were broadly cross-reactive within serogroups. Post-immune, but not pre-immune, sera bound to intact gonococci, induced deposition of complement components C3 and C9 onto gonococcal membranes and increased association with and activation of human neutrophils. Gonococci were not killed in bactericidal assays, and there was no phagocytic killing with gonococci opsonized with recombinant antisera. Lack of killing in bactericidal assays was not caused by the presence of blocking antibodies to the outermembrane protein Rmp.
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  • 34
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The PilR protein of Pseudomonas aeruginosa is a transcriptional activator of the pilin gene and belongs to a two-component sensor–regulator family. PilR was overproduced by fusing pilR to the gene for the maltose-binding protein (malE), yielding a MalE–PilR hybrid protein. The plasmid with the malE–pilR fusion, when introduced into a non-piliated pilR mutant strain of P. aeruginosa, restored piliation, indicating that the hybrid protein retains PilR function in vivo. The MalE-PilR protein was purified from Escherichia coli and used in a series of DNA-binding studies. A specific pilin promoter-binding activity of MalE-PilR was observed in a gef retardation assay. Subsequent DNase I footprinting analysis revealed a 40bp PilR-binding site located at the −120 to −80 region, relative to the transcriptional start site of the pilin gene. This PilR-binding region consists of a nine-base sequence and three consensus sequences of 5-(N)4–6C/GTGTC-3′, in a tandem array in which the first 7–9 bp are bound by the PilR on the non-Goding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PilR binds of sequences complementary to the two middle consensus sequences of the non-coding strand. A sequence similar to the NifA recognition site (5-TGT-(N)11-ACA-3′) is also found within the PilR-binding region. Deletion analysis and disruption of the individual consensus PilR-binding sequences by site-directed mutagenesis revealed that all four PilRbinding sites are absolutely required for the PilS/PilR-mediated pilin gene expression. The presence of four PilR-binding repeat sequences suggests that PilR protein may bind co-operatively or as a multimer.
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  • 35
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The dipeptide permease (Dpp) of Escherichia coli transports peptides consisting of two or three L-amino acids. The periplasmic dipeptide-binding protein (DBP), encoded by the dppA gene, also serves as a chemoreceptor. We sequenced the dpp locus, which comprises an operon of five genes, dppABCDE. Its organization is the same as the oligopeptide permease (opp) operon of Salmonella typhimurium and the spo0K operon of Bacillus subtilis. The dpp genes are also closely related to the hbpA gene, which encodes a haem-hinding lipoprotein, and four other genes in an unlinked operon of unknown function in Haemophilus influenzae. Each Dpp protein has an Opp, Spo0K and H. influenzae homologue. Transcription of the dpp operon initiates 165 bases upstream of the predicted dppA start codon. The start site for transcription is preceded by potential −35 and −10 regions of a σ70 promoter. During exponential growth in Luria-Bertani (LB) broth, the level of dpp mRNA increases in two steps, one between A590 0.2 and 0.4 and one between A590 0.7 and 1.0. The 310 nucleotides between dppA and dppB include a RIP (repetitive IHF-binding palindromic) element, whose deletion from a multi-copy plasmid causes fivefold and 10-fold reductions in the levels of upstream and downstream dpp mRNA, respectively.
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  • 36
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Interactions of Opc-expressing Neisseria meningitidis with polarized and non-polarized human umbilical vein endothelial cells (Huvecs) were investigated. Metabolic inhibitors and cytochalasin D treatment showed that host cellular and cytoskeletal functions were important for Opc-expressing bacterial association with Huvecs at the apical surface. In addition, this interaction required the presence of serum in the incubation medium whilst association with nonpolarized cells did not require serum. Pre-exposure of Opc-expressing bacteria to serum was sufficient to increase the number of bacterial interactions at the apical surface; B306, a monoclonal antibody (mAb) against Opc, inhibited these interactions, suggesting that Ope binds to serum factor(s) and this in turn increases adherence to Huvecs. The receptors involved in this ‘sandwich’ adherence belong to the integrin family since the interaction was inhibited by peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD) and the tetrapeptide RGDS (but not the peptide RGES) was inhibitory. Non-polarized cells appeared to expose receptors/sites that bound to Opc-expressing bacteria directly, did not require serum factors and were not inhibited by RGD-containing peptides. Serum-dependent interactions of Opc-expressing bacteria to apical surface was inhibited significantly by severai mAbs against avβ3 integrins. Some mAbs against α5 and β1 caused partial inhibition; antibodies that did not block the function of β1 integrins or the mAbs against α2 integrins were not inhibitory to bacterial interactions with Huvecs. Purified vitronectin supported adherence of Opc-expressing bacteria to Huvecs but not of Opc-bacteria. These interactions were inhibited by mAb B306 against Opc, by RGDS peptides as well as by blocking antibodies directed against αvβ-3 but not antibodies against other integrins. These data suggest that a sequence of molecular events resulting in trimolecular complexes at the endothelial surface may drive neisserial invasion of Huvesc. The expression of Opc appears to enable bacteria to utilize the normal signal-transduction mechanism of host cells via ligands in sera that adhere to endothelial cell integrins.
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  • 37
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 14 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
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  • 38
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The effects of defined mutations In the lipopolysaccharide (LPS) and the outer membrane protein OmpA of the recipient cell on mating-pair formation in liquid media by the transfer systems of the F-Iike plasmids pOX38 (F), ColB2 and R100-1 were investigated. Transfer of all three plasmids was affected differently by mutations in the rfa (LPS) locus of the recipient cell, the F plasmid being most sensitive to mutations that affected rfaP gene expression which is responslbie for the addition of pyrophosphorylethanolamine (PPEA) to heptose I of the inner core of the LPS. CoIB2 transfer was more strongly affected by mutations in the heptose II-heptose III region of the LPS (rfaF) whereas R100-1 was not strongly affected by any of the rfa mutations tested. ompA but not rfa mutations further decreased the mating efficiency of an F plasmid carrying a mutation in the mating-pair stabilization protein TraN. An F derivative with a chloramphenicol acetyltransferase (CAT) cassette interrupting the traA pilin gene was constructed and pilin genes from F-like plasmids (F, ColB2, R100-1) were used to complement this mutation. Unexpectediy, the results suggested that the differences in the pilin sequences were not responsible for recognizing specific groups in the LPS, OmpA or the TraT surface exclusion protein. Other corroborating evidence is presented suggesting the presence of an adhesin at the F pilus tip.
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  • 39
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra+ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.
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  • 40
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp. israelensis and the PG-14 isolate of B. thuringiensis subsp. morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which It is composed (CytA, CrylVA, B, and D). This high toxicity is postulated to be due to synergistic interactions among parasporal proteins. However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae. In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CrylVD toxins were cloned and expressed in acrystalliferous B. thuringiensis subsp. israelensis cells using the shuttle vector pHT3101. CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bio-assays against first instars of Aedes aegypti. The LC50 for the CytA inclusion was 60 ng ml−1, whereas the LC50 for the CryIVD was 85ng ml−1 In comparison, the LC50s for different combinations of CytA and CrylVD inclusions ranged from 12–15 ng ml−1, 4–5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins. These results suggest that the high toxicity of the wild-type parasporal bodies of B. thuringiensis subspp. israelensis and morrisoni Is due to synergism among three or four of their major proteins.
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  • 41
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A new locus required for type 4 pilus biogenesis by Pseudomonas aeruginosa has been identified. A pilE mutant, designated MJ-6, was broadly resistant to pili-specific phages and unable to translocate across solid surfaces by the pilus-dependent mechanism of twitching motility (Twt−). Immunoblot analysis demonstrated that MJ-6 was devoid of pili (Pil−) but was unaffected in the production of unassembled pilin pools. Genetic studies aimed at localizing the pilE mutation on the P. aeruginosa PAO chromosome demonstrated a strong co-linkage between MJ-6 phage resistance and the proB marker located at 71 min. Cloning of the pilE gene was facilitated by the isolation and identification of a proB+-containing plasmid from a PAO1 cosmid library. Upon introduction of the PA01 proB+ cosmid clone into MJ-6, sensitivity to pili-specific phage, twitching motility and pilus production were restored. The nucleotide sequence of a 1 kb Eco RV-Clal fragment containing the pilE region revealed a single complete open reading frame with characteristic P. aeruginosa codon bias. PilE, a protein with a molecular weight of 15278, showed significant sequence identity to the pilin precursors of P. aeruginosa and to other type 4 prepilin proteins. The region of highest homology was localized to the N-terminal 40 amino acid residues. The putative PilE N-terminus contained a seven-residue basic leader sequence followed by a consensus cleavage site for prepilin pep-tidase and a largely hydrophobic region which contained tyrosine residues (Tyr-24 and Tyr-27) previously implicated in maintaining pilin subunit-subunit interactions. The requirement of PilE in pilus biogenesis was confirmed by demonstrating that chromosomal pilE insertion mutants were pilus- and twitching-motility deficient.
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  • 42
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 13 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Deletion mutants of R100-1 were constructed by classical methods to remove various segments of the traM open reading frame, pTraM-binding sites and the traM promoters. Complementation tests showed that traM was efficiently complemented only when the frans-acting fragment contained both the complete traM gene and the adjacent traJ promoter and leader sequences. The conclusion is that traM and traJ constitute a complex operon. A deletion mutant lacking all of the fraJ gene, and one containing a frameshifting traM deletion, retained the ability to transfer at a low level, thereby showing that neither pTraM nor pTraJ is absolutely essential for transfer.
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  • 43
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Threonine is found at the third position of the second α-helix in the helix-turn-helix motifs of most bacterial DNA-binding proteins. To investigate the role of this conserved residue in Escherichia coli Trp repressor function, plasmids encoding mutant Trp repressers with each of the 19 amino acid changes of Thr-81 were made by site-directed mutagenesis. All 19 changes decrease the activity of Trp holorepressor, indicating that the Thr-81 side-chain is critical for TrpR function. Three mutant repressors, Ser-81, Lys-81 and Arg-81, retain partial DNA-binding activity and inhibit transcription from the wild-type trp promoter/operator complex; challenge-phage assays show that Ser-81 and Lys-81 holorepressors have altered DNA-binding specificities. The side-chain of Thr-81 may make direct contacts with base pairs 4 and 3 of the trp operator, consistent with the nuclear magnetic resonance solution structures of the holorepressoroperator complex.
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  • 44
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 13 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The distribution, characterization and function of the tcpA gene was investigated in Vibrio cholerae O1 strains of the El Tor biotype and in a newly emergent non-O1 strain classified as serogroup O139. The V. cholerae tcpA gene from the classical biotype strain O395 was used as a probe to identify a clone carrying the tcpA gene from the El Tor biotype strain E7946. The sequence of the E7946 tcpA gene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395. The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge. In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion in tcpA. This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotype tcpA mutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains. The tcpA analysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non-O1 strain known to cause epidemic cholera, an O139 V. cholerae isolate from the current widespread Asian epidemic. These strains were shown to carry tcpA with a sequence identical to E7946. These results provide further evidence that the newly emergent non-O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP-associated antigens. Therefore, there appear to be only two related sequences associated with TCP pilin required for colonization by all strains responsible for epidemic cholera, one primary sequence associated with classical strains and one for El Tor strains and the recent O139 derivative. A diagnostic correlation between the presence of tcpA and the V. cholerae to colonize and cause clinical is now extended to strains of both O1 and non-O1 serotypes.
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  • 45
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Complementary chromatic adaptation is a mechanism by which some cyanobacteria that are able to synthesize phycoerythrin can adapt their pigment (phycobili-protein) content to the incident wavelengths of the light. In Calothrix sp. PCC 7601 it concerns phycoerythrin (cpe operon), synthesized under green light, and phycocyanin-2 (cpc2 operon), expressed under red light, and involves transcriptional controls. With cell-free extracts from Calothrix sp. PCC 7601 grown under various light regimes, a protein designated RcaD was found by gel retardation experiments to specifically bind to the cpc2 promoter region and to be present only in red-light-grown cells. This protein was partially purified and its binding activity was shown to be sensitive to an alkaline phosphatase treatment. RcaO can protect two regions of the cpc2 promoter sequence against degradation by DNase I. Because its activity is detected only under the conditions required for cpc2 expression, we propose that RcaD is a positive effector of transcription.
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  • 46
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: All Haemophilus influenzae strains have an absolute requirement for exogenously supplied haem for aerobic growth. A majority of strains of H. influenzae type b (Hib) produce a 100 kDa protein which binds haem: haemopexin complexes. This 100 kDa haem:haemopexin binding protein, designated HxuA, was originally detected on the Hib cell surface. Monoclonal antibody (mAb)-based analyses revealed that the HxuA protein was also present in soluble form in Hib culture supernatants. This soluble HxuA protein exhibited haem:haemopexin-binding activity in a direct binding assay. Nucleotide sequence analysis of the hxuA gene from Hib strain DL42, together with N-terminal amino acid analysis of HxuA protein purified from Hib culture supernatant, revealed that this protein was synthesized as a 101 kDa precursor with a leader peptide that was removed to yield a 99kDa protein. Southern blot analysis of chromosomal DNA from four Hib and four non-typeable H. influenzae (NTHI) strains detected the presence of a single band in each strain that hybridized a Hib hxuA gene probe. Subsequent analysis of these NTHI strains showed that all four strains released into culture supernatant a haem:haemopexin-binding protein that migrated in SDS-PAGE at a rate similar or identical to that of the Hib HxuA protein. A Hib hxuA mutant was used to screen an NTHI genomic DNA library and an NTHI gene was cloned that complemented the mutation in this Hib strain. Nucleotide sequence analysis of this NTHI gene revealed that it encoded a protein with 87% identity to the Hib HxuA protein. The expression of HxuA by both Hib and NTHI strains indicates that this particular haem acquisition system is conserved among H. influenzae strains.
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  • 47
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: In order to produce a successful infection, Neisseria gonorrhoeae (GC) must attach to and invade mucosal epithelial cells. To identify GC gene products involved in this early interaction with host cells we constructed a gene bank derived from a clinical isolate of GC, and isolated a clone which had the capacity to adhere to the human endometrial adenocarcinoma tissue-culture line HEC-1-B. The cloned sequence was identified as a member of the opa gene family whose protein products have been associated with virulence. The GC chromosome contains numerous variant opa genes which, in MS11, are designated opaA-K. Previous work showed that expression of opaC confers a highly invasive phenotype upon strain MS11. When our cloned opa gene was mutated and returned to the GC MS11A chromosome by transformation and homologous recombination, we isolated one transformation that was significantly reduced in its invasive capacity. The locus mutated in this transformant was identified as opaH. Our resuits indicate that invasive-ness of GC for human epithelail cells can be determined by more than one opa gene in strain MS11 A.
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  • 48
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of Mr 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.
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  • 49
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: To distinguish between a gradual or an abrupt movement of the Escherichia coli nucleoid during partitioning we determined the distances between nucleoid borders and cell poles. Measurements were performed on fixed but hydrated cells and on living cells growing in steady state. The distance between nucleoid outer border and cell pole remained constant in cells with either one or two nucleoids. Thus the nucleoid outer borders moved gradually during the partition process. To study partitioning during recovery from protein-synthesis inhibition cells were treated with chloramphenicol. After growth resumption, cells and nucleoids first elongated before partitioning occurred. Again, no indication of a rapid displacement of the nucleoid to one-quarter and three-quarter positions in the cell was observed.
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  • 50
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Escherichia coli arginine repressor (ArgR) is an l-arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.
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  • 51
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The human oral bacterium Streptococcus gordonii expresses, on the cell surface, two antigenically related high-molecular-mass polypeptides denoted CshA and CshB, encoded by genes at separate chromosomal loci. The precursor form of CshA is composed of four distinct segments: (i) a 41-amino-acid residue leader peptide, (ii) W-terminal 42–878 residues, (iii) residues 879–2417 comprising 13 repeat blocks of 101 amino acid residues and three shorter blocks, and (iv) a C-terminal anchor domain similar to those present in some other Gram-positive bacterial cell-wall polypeptides. Insertional mutations within cshA reduced both cell-surface hydrophobicity and ability to adhere to oral Actinomyces naeslundii. Insertional mutations in cshB had less effect on hydrophobicity and coadherence. However, expression of both polypeptides was found to be necessary for streptococci to colonize the murine oral cavity.
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  • 52
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The serine and aspartate chemosensory receptors (Tsr and Tar) of Escherichia coli have two membrane-spanning regions TM1 and TM2. To investigate their roles in transmembrane signalling, we constructed two chimeric receptors from Tsr and Tar with heterologous combinations of TM1 and TM2: the N-terminus of one receptor, including TM1 and the periplasmic domain, was fused to the C-terminus of the other, beginning with TM2. Both of the chimeric receptor genes rescued the chemotactic defect of a receptorless E. coli strain, indicating that the chimeric receptors are functional. Their apparent affinities for the specific ligands were the same as those of Tsr or Tar. Therefore, as far as transmembrane signalling abilities are concerned, the TW2 regions of Tsr and Tar are interchangeable, suggesting that sequence-specific interaction between TM1 and TM2 may not be required for the signal transmission across the membrane.The cells expressing either of the chimeric receptors, however, showed ‘smooth’, biased, basal swimming patterns. Moreover, they adapted quickly after stimulation with the repellent glycerol. This rapid adaptation was observed even in the methyltransferase-defective strain. Therefore, exchange of TM2 might impose structural constraints on the chimeric receptors that stabilize conformations which elicit smooth swimming.
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  • 53
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 14 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A mutant Escherichia coli that transforms minichromosomes with high efficiency in the absence of Dam methylation has been Isolated and the mutation mapped to 16.25 min on the E. coli map. The mutant strain containing seqA2 is defective for growth in rich medium but not in minimal medium. A similar mutation In this gene, named seqA1, has also been isolated.Here we show that the product of the seqA gene, SeqA, normally acts as an inhibitor of chromosomal initiation. In the seqA2-containing mutant, the frequency of initiation increases by a factor of three. Introduction of the wild-type seqA gene on a low-copy plasmid suppresses the cold sensitivity of a dnaAcos mutant known to overinitiate at temperatures below 39°C. In addition, the seqA2 mutation is a suppressor of several dnaA (Ts) alleles.The seqA2 mutant overinitiates replication from oriC and displays the asynchronous initiation phenotype. Also the seqA2 mutant has an elevated level of DnaA protein (twofold).The introduction of minichromosomes or a low-copy-number plasmid carrying five DnaA-boxes from the oriC region increases the growth rate of the seqA2 mutant in rich medium to the wild-type level, reduces overinitiation but does not restore synchrony. We propose that the role of SeqA is to limit the activity level of the E. coli regulator of chromosome initiation, DnaA.
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  • 54
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 14 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The mechanism of iron utilization from transferrin has been most extensively characterized in the pathogenic Neisseria species and Haemophilus species. Two transferrin-binding proteins, Tbp1 and Tbp2, have been identified in these pathogens and are thought to be components of the transferrin receptor. Tbp1 appears to be an integral, TonB-dependent outer membrane protein while Tbp2, a lipoprotein, may be peripherally associated with the outer membrane. The relative contribution of each of these proteins to transferrin binding and utilization is discussed and a model of iron uptake from transferrin is presented. Sequence comparisons of the genes encoding neisserial transferrin-binding proteins suggest that they are probably under positive selection for variation and may have resulted from inter-species genetic exchange.
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  • 55
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Escherichia coli DnaK, DnaJ and GrpE are required for renaturation of heat-inactivated λ CI857 repressor (Gaitanaris et al., 1990). Here we demonstrate that in addition to the above three proteins, GroEL and GroES are necessary for the CI857 repressor to acquire full activity at the permissive temperature. Although full-length soluble repressor is present at normal amounts, the protein has reduced specific activity and migrates abnormally on native gels. To determine where the different chaperones act in protein folding, we identified their cellular locations. DnaK and DnaJ are associated with nascent polypeptide chains in translating ribosomes. In contrast, GroEL, although it is transiently associated with newly synthesized proteins, is absent from the ribosomes. This suggests that DnaK and DnaJ ptay an early role in protein maturation, whereas GroEL acts at a later stage.
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  • 56
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 14 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Haem O and/or haem A are specifically synthesized for the haem-copper respiratory oxidases. A 17-carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen-reduction site. The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis. Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase). It has recently been suggested that the ctaA gene adjacent to the ctaB-ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase). In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.
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  • 57
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Pyelonephritic isolates of Escherichia coli commonly express P-pili, which mediate bacterial attachment to glycolipids on epithelial cell surfaces. Three classes of P-pili have been defined, based on varying specificity for galabiose-containing glycolipids. Variation in adhesive capacity is correlated with a shift in preferred host, suggesting that host tropism depends largely on detailed specificity for the globoseries glycolipids. In this study we examined the importance of the PapG adhesin in determining receptor specificity. Translational fusions were constructed between the ammo-terminus of the PapG adhesin from each of the three pilus classes and a reporter protein. The binding specificity of the purified fusion proteins in vitro was identical to that seen with whole bacteria. Adherence of intact bacteria to cultured kidney cells was markedly reduced by a monoclonal antibody specific for the Class III adhesin (previously denoted PrsG), confirming the importance of the ammo-terminus of PapG in mediating attachment to a receptor when presented on the eukaryotic cell surface. These results suggest that the detailed receptor specificity resides solely within the amino-terminus of the PapG adhesin and is independent of the complex pilus architecture.
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  • 58
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Most of the genes required for the conjugative transfer of DNA are encoded by the 33 kb transfer (tra) operon of F-like conjugative plasmids. Transcription of the tra operon is positively regulated by the TraJ transcriptional activator which, in turn, is negatively regulated by the FinOP fertility inhibition system. The FinOP system consists of an antisense RNA, FinP, and a 21.2 kDa protein, FinO, which together inhibit TraJ expression. Previously, it has been demonstrated that FinO increases the in vivo stability of the FinP RNA in the absence of the traJ mRNA target. Using electrophoretic mobility shift assays, we have shown that FinO is an RNA-binding protein that binds to one of the two stem-loops in FinP and to its complementary structure in traJ mRNA. This interaction presumably protects FinP RNA from degradation in vivo and increases the rate of formation of the FinP- traJ mRNA duplex fivefold. Thus, TraJ expression appears to be influenced by a unique RNA-protein interaction that precedes duplex formation between the FinP anti-sense RNA and its target traJ mRNA.
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  • 59
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Retrons are genetic elements that encode multicopy single-stranded DNAs called msONAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single-stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem-loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem-loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to seqestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.
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  • 60
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Spo0A is a phosphorylation-activated transcription factor of Bacillus subtilis. It is a member of the response regulator super-family of bacterial signal transduction proteins and controls many of the changes in gene expression that occur during the transition into stationary phase and during the initiation of speculation. To identify the domains of Spo0A most critical for determining its structural and functional features, presumptive homologues of the spo0A gene were characterized in a collection of eight Bacillus species and six Clostridium species representing phylogenetically diverse members of these genera. An alignment of the partial or complete DNA sequences of these homologues revealed three regions of especially high conservation in the effector domain. We speculate that the most highly conserved of these corresponds to the recognition helix of a putative helix-turn-helix motif, and, therefore, represents the actual DNA-contacting surface of the protein. In the case of homologues identified in Bacillus anthracis and Clostridium acetobutylicum and retrieved by polymerase chain reaction amplification, we confirmed by gene-disruption analysis that the homologue actually is required for initiation of sporulation. Apparent homologues of the B. subtilis spolVB gene were also discovered immediately upstream from the spo0A homologues in all Bacillus and Clostridium species examined. The discovery of homologues of B. subtilis sporulation genes in these diverse species implies that the gene products required for specifying pathways of sporulation-specific gene activation and for determining key morphogenetic changes may be highly conserved and suggests that an approach similar to that undertaken here might be used as a general strategy to retrieve and compare their gene sequences. Exhaustive efforts to detect a spo0A-like gene in non-endospore formers, including close relatives of Bacillus such as Listeria and Staphylococcus, were uniformly unsuccessful, suggesting that regulation of gene activity during the transition into stationary phase mediated by Spo0A-like proteins may be exclusive to the endospore-forming bacteria.
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  • 61
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Curli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue-type plasminogen activator (t-PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t-PA with high affinity (1 × 108 to 2 × 108 M-1). The binding of plasminogen and t-PA to curli-expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli-expressing E. coli was readily converted to plasmin by t-PA; both plasmin and t-PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.
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  • 62
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    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 14 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The amount of the rate-limiting replication initiator protein RepR of plasmid p1P501 is negatively controlled by an antisense RNA (RNAIII) and a dispensable protein (CopR). Deletions or mutations in either component cause a 10-20-fold copy number increase. RNAIII induces transcription attenuation of the repR mRNA: the mode of CopR action remained unclear. To test the function of CopR, transcriptional fusions of promoters pI, pII and pIII with lacZ were integrated into the Bacillus subtilis chromosome. CopR and/or RepR were supplied in trans, and LacZ synthesis measured. The results show that CopR represses the repR promoter pII. Neither CopR nor RepR autoregulate their promoters. Gel mobility shift assays indicate that CopR binds to a 44 bp DNA fragment comprising the inverted repeat upstream of pII.
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  • 63
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Campylobacter fetus utilizes paracrystalline surface (S-) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full-length S-layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full-length S-layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 × 10-2. Immunoblots of survivors showed that phenotypic reversion involving high-level production of full-length (98, 127 or 149 kDa) S-layer proteins had occurred. Revertants were serum resistant but caused approximately 10-fold less bacteraemia in orally challenged mice than did the wild-type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high-frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.
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  • 64
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: pAMβ1 is a low-copy-number, promiscuous plasmid from Gram-positive bacteria that replicates by a unidirectional theta-type mode. Its replication is initiated by an original mechanism, involving the positive rate-limiting RepE protein. Here we show that the pAMβ1-encoded CopF protein is involved in negative regulation of the plasmid copy number. CopF represses -10-fold the transcription initiated at the promoter of the repE gene and binds to a 31 bp segment which is located immediately upstream of the -35 box of the repE promoter. We propose that CopF inhibits initiation of transcription at the repE promoter by binding to its operator.
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  • 65
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    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 14 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Development of the Escherichia coli cell division site was studied in wild-type cells and in non-septate filaments of ftsZ null and ftsZTs mutant cells. Localized regions of plasmolysis were used as markers for the positions of annular structures that are thought to be related to the periseptal annuli that flank the ingrowing septum during cytokinesis. The results show that these structures are localized at potential division sites in non-septate filaments of FtsZ- cells, contrary to previous reports. The positions of the structures along the long axis of the cells in both wild-type cells and FtsZ- filaments were unaffected by the presence of plasmolysis bays at the cell poles. These results do not agree with a previous suggestion that the apparent association of plasmolysis bays with future division sites was artefactual. They support the view that division sites begin to differentiate before the initiation of septal ingrowth and that plasmolysis bays and the annular attachments that define them, mark the locations of these early events in the division process.
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  • 66
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    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 14 (1994), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Early changes at cell-division sites were studied in non-septate filaments induced by growth of ftsATs mutant cells under non-permissive conditions. The positions of localized regions of plasmolysis were used as markers for the locations of partial and complete annular structures that are thought to be precursors of the periseptal annuli that flank the septum during cytokinesis. The results confirmed that these structures were localized at potential division sites and suggested a model in which older division sites play a role in the generation of new sites for future use, with each older site being used only once for this purpose. The results also suggest that the details of division-site development can profitably be studied in cells in which early events in the differentiation process are uncoupled from the septation event.
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  • 67
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Monoclonal antibodies were raised against a fusion between the Escherichia coli maltose-binding protein and LciA, the immunity protein that protects Lactococcus lactis against the effects of the bacteriocin lactococcin A. One of the antibodies directed against the LciA moiety of the fusion protein was used to locate the immunity protein in the L. lactis producer cell. LciA was present in the cytosolic. the membrane-associated, and the membrane fractions in roughly equal amounts, irrespective of the production by the cells of lactococcin A.The monoclonal antibody specifically reacted with right-side-out vesicles obtained from a strain producing the immunity protein. It did not react with inside-out vesicles of the same strain, or with right-side-out vesicles obtained from a strain producing both LciA and lactococcin A. Also, externally added lactococcin A blocked the interaction between the antibody and right-side-out vesicles obtained from a strain producing only LciA.The epitope in LciA was localized between amino acid residues 60 and 80. As the epitope could be removed from right-side-out vesicles by proteinase K, it is located at the outside of the cell.The immunity protein contains a putative a-amphiphilic helix from residue 29 to 47. A model is proposed in which this helix is thought to traverse the membrane in such a way that the C-terminal part of the protein, containing the epitope, is on the outside of the cell.Vesicle-fusion studies together with leucine-uptake experiments suggest that the immunity protein interacts with the putative receptor for lactococcin A, thus preventing pore formation by the bacteriocin.
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  • 68
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The rfbkpO1 gene cluster of Klebsiella pneumoniae O1 directs synthesis of the D-galactan I component of the lipopolysaccharide O-antigen. The first two genes in the rfbkpO1cluster encode RrfbkpO1and RfbBKpO1, with predicted sizes of 29.5 or 30.0 kDa and 27.4 kDa, respectively. RfbBKpO1 contains a consensus ATP-binding domain and shares homology with several proteins which function as ATP-binding components of cell surface polysaccharide transporters. RfbAKpO1 is predicted to be an integral membrane protein with five putative membrane-spanning domains and its transmembrane topology was confirmed by TnphoA mutagenesis. The hydropathy plot of RfbAKpO1 resembles KpsM, the transcytoplasmic membrane component of the capsular polysaccharide transporter from Escherichia coli K-1 and K-5. These relationships suggest that RfbAKpO1 and RfbBKpO1 belong to a family of two-component ABC (ATP-binding cassette) transporters. E. coli K-12 containing a plasmid carrying an rfbKpO1 gene cluster deleted in rfbAKpO1 and rfbBKpO1 expresses rough lipopolysaccharide molecules on its surface and accumulates cytoplasmic O-antigen. When RfbAKpO1 and RfbBKpO1 are supplied in trans by a compatible plasmid, O-polysaccharide transport is restored and smooth D-galactan l-substituted lipopolysaccharide is produced. RfbAKpO1 and RfbBKpO1 are, therefore, proposed to constitute a system required for transport of D-galactan I across the cytoplasmic membrane, where RfbAKpO1 represents the membrane-spanning translocator and RfbBKpO1 couples the energy of ATP hydrolysis to the transport process.
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  • 69
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The stationary-phase-specific sigma factor σS (RpoS/KatF) is required for Escherichia coli to induce expression of fibronectin-binding curli organelles upon reaching stationary phase. We show that the csgA gene which encodes the curlin subunit protein belongs to a dicistronic operon, csgBA. The transcriptional start site of csgBA was determined and an AT-rich upstream activating sequence (UAS) required for transcriptional activation was identified. The pcsgBA promoter is not specific for σS since the same promoter sequence can be used by Eσ70in vivo in a strain lacking nucleoid-associated protein H-NS and σS. Transcription remained growth-phase induced and dependent upon the UAS in such a double mutant. Furthermore, we demonstrate that an additional operon, hdeAB, which is also dependent upon σS for transcription, can be transcribed by Eσ70in vivo in the absence of H-NS by utilizing the phdeAB promoter. Two other genes known to be under the control of σS for expression, bolA and katE, remained transcriptionally silent in the absence of H-NS. It is suggested that a subset of E. coli promoters can be recognized by both EσS and Eσ70in vivo but H-NS interacting with these sequences prevents formation of succesful transcription-initiation complexes with Eσ70.
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  • 70
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The structure, genomic organization and transcription of the gene encoding histone H2B in the protozoan parasite Trypanosoma cruzi have been studied. This gene consists of a 746-nucleotlde unit, tandemly repeated at least 18 times in each of two clusters. DNA probes corresponding to histones H2B and H3 hybridized to different chromosomes revealing that the genes coding for these two histones are not physically linked in the genome of T. cruzi. The primary transcription product of the H2B gene is processed by trans-splicing and polyadenylation. Inhibition of DNA synthesis with aphidicolin resulted in the reduction of histone H2B mRNA to undetectable levels in about two hours, suggesting that its abundance is regulated throughout the cell cycle as it occurs in other eukaryotes. in addition, a concomitant inhibition of translation by cycloheximide reverted this effect indicating that de novo protein synthesis is required for RNA instability. Histone mRNA abundance was dependent on the life-cycle stage of T. cruzi: abundant in amastigotes and epimastigotes, the dividing forms in the host cell and the insect vector, respectively, white undetected in trypomastigotes, the parasite's non-dividing life stage.
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  • 71
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Expression from both the Escherichia coli nir and nrf promoters is dependent on anaerobic induction by FNR but is further regulated by NarL and NarP in response to the presence of nitrite and nitrate in the growth medium. The nir promoter is activated by NarL in response to nitrate and nitrite and activated by NarP in response to nitrate but not nitrite. The effects of point mutations suggest that NarL and NarP both bind to the same target, which is a pair of heptamer sequences organized as an inverted repeat, centred 691/2 bp upstream of the transcript startpoint. The nrf promoter can be activated by either NarP or NarL in response to nitrite but is repressed by NarL in response to nitrate. Mutational analysis of the nrf promoter has been exploited to corroborate the location of the -10 hexamer and the FNR-binding site, and to find the sites essential for nitrite-dependent activation and nitrate-dependent repression. Optimal activation by NarP or NarL in response to nitrite requires an inverted pair of heptamer sequences, similar to that found at the nir promoter, but centred 741/2 bp upstream from the transcript start. NarL-dependent repression by nitrate is due to two heptamer sequences that flank the FNR-binding sequence. We conclude that NarL and NarP bind to the same heptamer sequences, but that the affinities for the two factors vary from site to site.
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  • 72
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Haemophilus influenzae represents a common cause of human disease and an important source of morbidity and mortality. Disease caused by this organism begins with colonization of the upper respiratory tract. Several studies indicate that H. influenzae is capable of binding to and entering cultured human cells, properties which are potentially of relevance to the process of colonization. In the present study, we isolated an H. influenzae gene designated hap, which is associated with the capacity for In vitro attachment and entry. Analysis of the derived amino acid sequence of hap demonstrated significant homology with the serine-type lgA1 proteases expressed by H. influenzae and Neisseria gonorrhoeae. It is notable that the hap product shares the catalytic domain of the lgA1 proteases and appears to be processed and secreted in an analogous manner. We speculate that the hap gene product is an important determinant of colonization, perhaps enabling the organism to evade the local immune response and thereby persist within the respiratory tract.
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  • 73
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The involvement of the RTX haemolysins (Apxl and ApxII) of the swine pathogen Actinobacillus pleuropneumoniae in virulence was investigated using haemolysin-deficient mutants constructed by a mini-Tn10 mutagenesis procedure. Two types of haemolysin mutant with single insertions of the transposon were obtained from a serotype 1 strain producing both ApxI and ApxII. One presented a complete loss of haemolytic activity because of the absence of ApxI and ApxII production. The other displayed weaker haemolysis than the wild type and produced only ApxII. The chromosomal regions flanking mini-Tn10 were cloned and sequenced. In the non-haemolytic mutant, the transposon had inserted in apxIB, a gene involved in the exportation of ApxI and ApxII toxins. The weakly haemolytic mutant resulted from the disruption of the structural gene for ApxI. Both mutations In the apxI operon were associated with a significant loss of virulence for mice and pigs, demonstrating that haemolysins are involved in A. pleuropneumoniae pathogenicity. The non-haemolytic mutant was apathogenic and the weakly haemolytic mutant retained some virulence for pigs, suggesting that both ApxI and ApxII are needed for full virulence.
    Type of Medium: Electronic Resource