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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 243 (2005), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We have developed an oligonucleotide-chip based assay for detection of 16S ribosomal PCR products from tick-borne bacteria. This chip contains 14 specific probes, which target variable regions of 16S rDNA of tick-borne bacteria including Borrellia spp., Rickettsia spp., Anaplasma spp., Coxiella burnetii and Francisella tularensis. The specificity of these probes was tested by hybridization of the chip with fluorescently labeled PCR products amplified from the genomic DNA of selected tick-borne bacteria. The assay was also tested for detection of tick-borne bacteria in single ticks.
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  • 2
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The response regulator proteins of two-component systems mediate many adaptations of bacteria to their ever-changing environment. Most response regulators are transcription factors that alter the level of transcription of specific sets of genes. Activation of response regulators requires their phosphorylation on a conserved aspartate residue by a cognate sensor kinase. For this reason, expression of a recombinant response regulator in the absence of the requisite sensor kinase is expected to yield an unphosphorylated product in the inactive state. For Spo0A, the response regulator controlling sporulation in Bacillus subtilis however, we have found that a significant fraction of the purified recombinant protein is phosphorylated. This phosphorylated component is dimeric and binds to Spo0A recognition sequences in DNA. Treatment with the Spo0A-specific phosphatase, Spo0E, leads to dissociation of the dimers and loss of DNA binding. It is therefore necessary to pre-treat recombinant Spo0A preparations with the cognate phosphatase, to generate the fully inactive state of the molecule.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 185 (2000), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Spo0A is a two domain response regulator, a key protein in the initiation of sporulation of Bacillus subtilis. This protein controls a number of changes in gene expression that occur during the transition from stationary phase to the onset of sporulation. The phosphorylated form of Spo0A influences the transcription of a specific set of genes. In addition to others, it represses abrB and activates spoIIA and spoIIE transcription. Although the N-terminal phosphoacceptor domain is well characterised, there is limited information on the C-terminal, DNA-binding domain. Comparisons of Spo0A homologues from a number of Bacillus and Clostridium species show that the C-terminal domain contains three highly conserved regions. In this study, we have investigated the influence of spo0A mutations mapping within the C-terminal domain on transcription from the abrB, spoIIA and spoIIE promoters using lacZ fusions. Our results indicate that described mutations can be part of signalling between N- and C-terminal domains of the protein. Also, the increased expression observed from the spoIIE promoter in some Spo0A mutants might result from a stabilising function of these mutations on the transcriptional apparatus utilising σA.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 175 (1999), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The molecular biological study of the obligate intracellular bacterium Coxiella burnetii is hampered because of the lack of an efficient DNA transformation system. We used expression of the green fluorescent protein (GFP) in addition to ampicillin resistance as a selection marker for detection of transformed C. burnetii cells. Fluorescent microscopy studies revealed that transformed C. burnetii cells can be detected easily inside the host cell line. A high level of GFP expression was reached with the strong Escherichia coli trc (trp/lac) promoter. The use of GFP not only provides a convenient marker for transformation of C. burnetii, but also allows detection of this obligate intracellular pathogen inside host eukaryotic cells. Possible applications for GFP in the study of host-pathogen interactions are discussed.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 172 (1999), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an Mr of 29 422 showed the greatest similarity to the previously identified sporulation sigma factor (σF) of Streptomyces coelicolor, and general stress response sigma factor (σB) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of −10 and −35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 153 (1997), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The Streptomyces aureofaciens sigF gene encodes a sigma factor. By integrative transformation, via double cross-over, a stable null mutant of sigF gene was obtained. This mutation appeared to have no obvious effect on vegetative growth, but affected the late stage of spore maturation. Microscopic examination showed that spores were deformed, and spore wall was thinner, compared with the wild-type spores. The spore pigment of sigF mutant was green, compared to wild-type grey-pink spore pigmentation. The plasmid-born wild-type sigF gene complemented the mutation after transformation of the mutant strain.
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  • 7
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Functioning of the spoIIE locus of Bacillus subtilis is required for formation of a normal polar septum during sporulation and for activation of the transcription factor σF, which directs early forespore-specific gene expression. We have determined the DNA sequence of the wild type and several mutant alleles of the spoIIE gene of B. subtilis and sequenced a substantial portion of its presumptive homologue in Bacillus megaterium. We show that the spoIIE locus encodes a single large protein with a predicted molecular mass of 92 kDa. Each of five point-mutation alleles, which have traditionally defined the locus, and two transposon-generated mutations were shown to fall within the coding sequence for the 92 kDa gene product or within sequences expected to be required for its expression. The amino-terminal portion of the predicted SpoIIE gene product, comprising approximately 40% of the protein, is extremely hydrophobic and is expected to contain up to 12 membrane-spanning segments. The remainder of the protein contains no hydrophobic segments long enough to span a lipid bilayer and is therefore presumed to comprise one or more globular, aqueous-phase exposed domains. An in-frame fusion joining the 3′ end of the B. megaterium spoIIE coding sequence to the 5′ end of gfp, a gene encoding the green fluorescent protein (GFP) of Aquorea victoria, resulted in a strong, sporulation-specific fluorescent signal localized to the sites of sporulation septum assembly. We speculate that SpoIIE plays a role in assembling the sporulation septum, perhaps determining the special properties of the structure that permit intercompartment signalling during development.
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 55 (2005), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Sporulation in the Gram-positive bacterium, Bacillus subtilis, has been used as an excellent model system to study cell differentiation for almost half a century. This research has given us a detailed picture of the genetic, physiological and biochemical mechanisms that allow bacteria to survive harsh environmental conditions by forming highly robust spores. Although many basic aspects of this process are now understood in great detail, including the crystal and NMR structures of some of the key proteins and their complexes, bacterial sporulation still continues to be a highly attractive model for studying various cell processes at a molecular level. There are several reasons for such scientific interest. First, some of the complex steps in sporulation are not fully understood and/or are only described by ‘controversial’ models. Second, intensive research on unicellular development of a single microorganism, B. subtilis, left us largely unaware of the multitude of diverse sporulation mechanisms in many other Gram-positive endospore and exospore formers. This diversity would likely be increased if we were to include sporulation processes in the Gram-negative spore formers. Spore formers have great potential in applied research. They have been used for many years as biodosimeters and as natural insecticides, exploited in the industrial production of enzymes, antibiotics, used as probiotics and, more, exploited as possible vectors for drug delivery, vaccine antigens and other immunomodulating molecules. This report describes these and other aspects of current fundamental and applied spore research that were presented at European Spores Conference held in Smolenice Castle, Slovakia, June 2004.
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  • 9
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Sporulation in Bacillus involves the induction of scores of genes in a temporally and spatially co-ordinated programme of cell development. Its initiation is under the control of an expanded two-component signal transduction system termed a phosphorelay. The master control element in the decision to sporulate is the response regulator, Spo0A, which comprises a receiver or phosphoacceptor domain and an effector or transcription activation domain. The receiver domain of Spo0A shares sequence similarity with numerous response regulators, and its structure has been determined in phosphorylated and unphosphorylated forms. However, the effector domain (C-Spo0A) has no detectable sequence similarity to any other protein, and this lack of structural information is an obstacle to understanding how DNA binding and transcription activation are controlled by phosphorylation in Spo0A. Here, we report the crystal structure of C-Spo0A from Bacillus stearothermophilus revealing a single α-helical domain comprising six α-helices in an unprecedented fold. The structure contains a helix–turn–helix as part of a three α-helical bundle reminiscent of the catabolite gene activator protein (CAP), suggesting a mechanism for DNA binding. The residues implicated in forming the σA-activating region clearly cluster in a flexible segment of the polypeptide on the opposite side of the structure from that predicted to interact with DNA. The structural results are discussed in the context of the rich array of existing mutational data.
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  • 10
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Making a spore in Bacillus subtilis requires the formation of two cells, the forespore and the mother cell, which follow dissimilar patterns of gene expression. Cell specificity is first established in the forespore under the control of the σF factor, which is itself activated through the action of the SpoIIE serine phosphatase, an enzyme targeted to the septum between the two cells. Deletion of the 10 transmembrane segments of the SpoIIE protein leads to random distribution of SpoIIE in the cytoplasm. Activation of σF is slightly delayed and less efficient than in wild type, but it remains restricted to the forespore in a large proportion of cells and the bacteria sporulate with 30% efficiency. Overexpression of the complete SpoIIE protein in a divIC mutant leads to significant σF activity, indicating that the septum requirement for activating σF can be bypassed. In contradiction to current models, we propose that genetic asymmetry is not created by unequal distribution of SpoIIE within the sporangium, but by exclusion of an inhibitor of SpoIIE from the forespore. This putative inhibitor would be a cytoplasmic molecule that interacts with SpoIIE and shuts off its phosphatase activity until it disappears specifically from the forespore.
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