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  • 1
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Target gene candidates of the seven extracytoplasmic function (ECF) sigma factors of Bacillus subtilis have been surveyed using DNA microarray analysis of mRNA extracted from cells grown in Luria–Bertani broth, in which an ECF sigma factor gene was placed under the control of the spac promoter on multicopy plasmid pDG148 and overexpressed. The number of target candidates for each of the sigma factors varied greatly, and a total of 278 genes were selected. Interestingly, the above target gene candidates shared only one gene out of 94 target genes of the general stress sigma B that have been reported in the literature thus far. Furthermore, lacZ-fusion experiments based on the results of DNA microarray analysis indicated that each ECF sigma factor directs transcription of its own operon, with the exception of sigZ. The DNA microarray data collected in this study are available at the KEGG Expression Database web site ().
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-LacI family of bacterial regulatory proteins, and the seryl-phos-phorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the ere in the first gene (gntR) of the gnt operon (credown), this operon contains another ere located in the promoter region (creup). A translational gntR-lacZ fusion expressed under the control of various combinations of wild-type and mutant credown and creup was integrated into the chromosomal amyE locus, and then catabolite repression of p-galac-tosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by creup was probably independent of catabolite repression exerted by credown; both creup and credown catabolite repression involved CcpA. Catabolite repression exerted by creup was independent of P-ser-HPr, and catabolite repression exerted by credown was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize creup, in contrast to its specific recognition of credown. However, CcpA complexed with glucose-6-phosphate specifically recognized creup as well as credown, but the physiological significance of this complexing is unknown.
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Catabolite repression of various Bacillus subtilis catabolic operons which carry a cis-acting catabolite-responsive element (ORE), such as the gnt operon, is mediated by CcpA, a protein belonging to the GalR-Lacl family of bacterial transcriptional repressors/activators, and the seryl-phosphorylated form of HPr, a phosphocarrier protein of the phosphoenol-pyruvate:sugar phosphotransferase system. Footprinting experiments revealed that the purified CcpA protein interacted with P-ser-HPr to cause specific protection of the gnt CRE against DNase I digestion. The specific recognition of the gnt CRE was confirmed by the results of footprinting experiments using mutant gnt CREs carrying one of the following base substitutions within the CRE consensus sequence: G to T at position +149 or C to Tat position +154 (+1 is the gnt transcription initiation nucleotide). The two mutant CREs causing a partial relief from catabolite repression were not protected by the CcpA/P-ser-HPr complex in footprinting experiments. Based on these and previous findings, we propose a molecular mechanism underlying catabolite repression in B. subtilis mediated by CcpA and P-ser-HPr.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respective cis-elements located upstream of the ilv-leu promoter. This operon is known to be under CcpA-dependent positive regulation. We have currently identified a catabolite-responsive element (cre) for this positive regulation (bases −96 to −82; +1 is the ilv-leu transcription initiation base) by means of DNase I-footprinting in vitro, and deletion and base-substitution analyses of cre. Under nitrogen-rich growth conditions in glucose-minimal medium supplemented with glutamine and amino acids, CcpA and CodY exerted positive and negative regulation of ilv-leu, respectively, but TnrA did not function. Moreover, CcpA and CodY were able to function without their counteracting regulation of each other, although the CcpA-dependent positive regulation did not overcome the CodY-dependent negative regulation. Furthermore, under nitrogen-limited conditions in glucose-minimal medium with glutamate as the sole nitrogen source, CcpA and TnrA exerted positive and negative regulation, respectively, but CodY did not function. This CcpA-dependent positive regulation occurred without the TnrA-dependent negative regulation. However, the TnrA-dependent negative regulation did not occur without the CcpA-dependent positive regulation, raising the possibility that this negative regulation might decrease the CcpA-dependent positive regulation. The physiological role of this elaborate transcription regulation of the B. subtilis ilv-leu operon in overall metabolic regulation in this organism is discussed.
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen-limited growth. In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome-wide screening for TnrA-regulated genes associated with a TnrA box. A combination of DNA microarray hybridization and a genome-wide search for TnrA boxes allowed us to find 36 TnrA-regulated transcription units associated with a putative TnrA box. Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF) possessed a common TnrA box. The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA. The TnrA targets detected in this study were nrgAB, pucJKLM, glnQHMP, nasDEF, oppABCDF, nasA, nasBCDEF and ywrD for positive regulation, and gltAB, pel, ywdIJK, yycCB, yttA, yxkC, ywlFG, yodF and alsT for negative regulation, nrgAB and gltAB being well-studied TnrA targets. It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets. The physiological role of the TnrA regulon is discussed.
    Type of Medium: Electronic Resource
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