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  • 1995-1999  (13)
  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 134 (1995), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract To facilitate purification of the two chaperonins GroES and GroEL encoded by the thermophilic Bacillus stearothermophilus, an Escherichia coli strain was constructed in which the groESL operon was replaced by that of B. stearothermophilus. This strain is perfectly viable, demonstrating that the B. stearothermophilus operon is functionally interchangeable with that of E. coli. To increase the amount of GroES, the groES gene was fused to an IPTG-inducible promoter. Both proteins GroES and GroEL were purified from E. coli using the standard protocol with some modifications. This method should be applicable in all cases where a foreign groE operon can substitute that of E. coli. A preliminary characterization of GroEL revealed that it has the same secondary structural elements as the E. coli homologue, but its thermodynamic stability is significantly increased.
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 19 (1996), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The induction of stress proteins is an important component of the adaptional network of a non-growing cell of Bacillus subtilis. A diverse range of stresses such as heat shock, salt stress, ethanol, starvation for oxygen or nutrients etc. induce the same set of proteins, called general stress proteins. Although the adaptive functions of these proteins are largely unknown, they are proposed to provide general and rather non-specific protection of the cell under these adverse conditions. In addition to these non-specific general stress proteins, all extracellular signals induce a set of specific stress proteins that may confer specific protection against a particular stress factor. In B. subtilis at least three different classes of heat-inducible genes can be defined by their common regulatory characteristics: Class I genes, as exemplified by the dnaK and groE operons, are most efficiently induced by heat stress. Their expression involves a σA-dependent promoter, an inverted repeat (called the CIRCE element) highly conserved among eubacteria, and probably a repressor interacting with the CIRCE element. The majority of general stress genes (class II, more than 40) are induced at σB-dependent promoters by different growth-inhibiting conditions. The activation of σB by stress or starvation is the crucial event in the induction of this large stress regulon. Only a few genes, including lonclpCclpP, and ftsH, can respond to different stress factors independently of σB or CIRCE (class III). Stress induction of these genes occurs at promoters presumably recognized by σA and probably involves additional regulatory elements which remain to be defined.
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  • 3
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Here we report on the construction of two integrative plasmids for Bacillus subtilis allowing in vitro construction of translational fusions. Both plasmids contain two cassettes in tandem: the bgaB gene encoding a heat-stable β-galactosidase devoid of its own regulatory sequences and the first two codons followed by a neomycin-resistance gene for selection in B. subtilis. Both cassettes are flanked by the 3′- and 5′-end of the amyE gene (encoding α-amylase) allowing integration of both cassettes at the amyE locus of the B. subtilis chromosome. For propagation in Escherichia coli, the plasmids contain the pBR322 origin of DNA replication and the β-lactamase-encoding gene. Whereas one vector needs a promoter, a Shine-Dalgarno sequence and the beginning of a gene fused in-frame to bgaB, the other one already carries a constitutive promoter. The versatility of the gene fusion vectors was demonstrated by the integration of the regulatory regions of the dnaK and the cat-86 genes. In the first case, heat-inducible expression was found, and by comparison with an operon fusion, it seems that the dnaK operon is regulated at both the transcriptional and the posttranscriptional level. In the second case, chloramphenicol-inducible regulation of the gene fusion could be demonstrated.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology reviews 23 (1999), S. 0 
    ISSN: 1574-6976
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The ftsH gene encodes an ATP- and Zn2+-dependent metalloprotease with a molecular mass of about 70 kDa. It was first identified in Escherichia coli where it is also designated hflB, tolZ or mrsC, and seems to be present in most if not all bacteria. The FtsH protein is anchored to the cytoplasmic membrane via two transmembrane regions in such a way that the very short amino- and the long carboxy-termini are exposed into the cytoplasm. FtsH is member of the AAA family (ATPases associated with a variety of cellular activities) which are characterized by a module of about 200 amino acid residues in length containing an ATP-binding site. In Escherichia coli, FtsH forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC. The E. coli enzyme is required for proteolytic degradation of some unstable proteins that include both soluble regulatory proteins such as σ32 (heat-shock sigma factor) and phage λ CII (transcriptional activator), and membrane proteins including uncomplexed forms of SecY (forms the translocon together with SecE and SecG) and the a subunit of the F0 complex of the H+-ATPase. Its activity can be modulated by the HflKC proteins, by another membrane protein designated YccA which can transiently associate with both the FtsH and the HflKC proteins, or by small peptides such as CIII encoded by phage λ (involved in lysogenization) or SpoVM (needed for sporulation) encoded by Bacillus subtilis. Besides being a protease, there is circumstantial evidence that FtsH also acts as a molecular chaperone. It influences protein assembly in and through the cytoplasmic membrane and associates with denatured alkaline phosphatase without degrading it. Therefore, FtsH may serve to maintain quality control of some cytoplasmic and membrane proteins. Such ATP-dependent proteases with intrinsic chaperone activity have been designated charonins.
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  • 5
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 15 (1995), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Bacillus subtilis contains at least three classes of heat-shock genes regulated by different mechanisms. We are studying class I heat-shock genes encoded by the operons dnaK and groE. These two operons are both expressed from a vegetative promoter, and their regulation involves a novel heat-shock element designated CIRCE. Here we show that induction of both operons results from enhanced synthesis of mRNA and is independent of de novo protein synthesis. To answer the question of whether dnaK is involved in the deregulation of the heat-shock response as reported for Escherichia coli, two different insertion mutations were isolated within the tetracistronic dnaK operon (orf39–grpE–dnaK–dnaJ). In one mutant a cat cassette was inserted at the beginning of orf39. Transcriptional analysis revealed that this mutation abolished expression of the whole operon. In contrast, the basal level of groE mRNA was significantly increased at 37°C, followed by a prolonged delay in the shut off after temperature upshift. These data point to a crucial role for the orf39 gene in the regulation of class I heat-shock genes. In the other mutant an internal 0.8 kb Bgl II fragment of dnaK was replaced by the cat cassette. In contrast to E. coli dnaK null mutants, the two B. subtilis dnaK operon mutants could grow within a temperature range from 16–52°C. At temperatures above 52°C, they failed to form colonies on agar plates, started to filament, and lost motility. Furthermore, the induction profile of the groE and dnaK operons was not impaired in the dnaK::cat mutant. The expression of the flagellin gene is influenced on both the transcriptional and post-transcriptional level. By Northern-blot analysis we previously showed that expression of the dnaK operon resulted in two mRNA species of 4.9 and 2.6 kb. Our mutational analysis suggests the formation of the 2.6 kb transcript as a processing product of the 4.9 kb species.
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  • 6
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 32 (1999), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The heptacistronic dnaK heat shock operon of Bacillus subtilis consists of the genes hrcA, grpE, dnaK, dnaJ, orf35, orf28 and orf50. It is controlled by the CIRCE/HrcA operator/repressor system and specifies three primary transcripts, two of which are processed into three different products. We have analysed the regulatory consequences of this complex transcriptional organization in detail. First, the seven genes were heat induced to different extents at the mRNA level and can be classified into three groups by their induction factors. This differential induction was also reflected at the protein level. Secondly, the cellular amounts of the proteins HrcA, DnaK and DnaJ in B. subtilis differed drastically both under non-heat shock conditions and after thermal upshock. Thirdly, Northern blot analyses demonstrated that an mRNA-processing reaction generating products of differential stabilities plays an essential role during the regulation of gene expression. A crucial factor determining the low stability of two transcripts is the presence of the CIRCE element at their 5′ ends. We demonstrate that CIRCE leads to the destabilization of mRNAs, but only if it is located in the immediate vicinity of a Shine–Dalgarno sequence. These results show that B. subtilis is using various, especially post-transcriptional, regulatory mechanisms to fine tune the expression of the individual genes of the heptacistronic dnaK operon.
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  • 7
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The ftsH gene of Bacillus subtilis has been identified as a general stress gene which is transiently induced after thermal or osmotic upshift. The FtsH protein exhibits 70.1% homology to FtsH of Escherichia coli which constitutes an essential ATP- and Zn2+-dependent protease anchored in the cytoplasmic membrane via two N-terminal transmembrane domains. This paper describes the isolation and functional characterization of an ftsH null mutant which was obtained by integration of a cat-cassette near the 5′ end of ftsH, thereby preventing the synthesis of FtsH protein. In contrast to the situation in E. coli, ftsH is dispensable in B. subtilis but results in a pleiotropic phenotype. While the mutant cells grew mostly as large filaments under physiological conditions, they turned out to be extremely sensitive to heat and salt stress. Although ftsH is necessary for adaptation to heat, it is not involved in the regulation of the heat-shock response. The induction profiles of representative genes of the CIRCE and sigma-B regulon and class III heat-shock genes lon and clpC were identical in the wild type and the ftsH null mutant. Furthermore, the ftsH knockout strain was unable to sporulate, and this failure was probably due to the absence of Spo0A protein which is essential for entry into the sporulation programme. In addition, secretion of bulk exoproteins was severely impaired in the ftsH null mutant after entry into stationary phase. The α-amylase and subtilisin activity in the supernatant was specifically tested. Whereas the activity of α-amylase increased after entry into stationary phase in both the wild type and the ftsH mutant strain, that of subtilisin encoded by aprE was prevented at the level of transcription in the mutant. Most of these results can be explained by the failure to synthesize appropriate amounts of Spo0A protein in the ftsH null mutant and point to ftsH as a developmental checkpoint
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  • 8
    Publication Date: 1999-01-01
    Print ISSN: 0168-6445
    Electronic ISSN: 1574-6976
    Topics: Biology
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  • 9
    Publication Date: 1995-12-01
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 10
    Publication Date: 1997-07-01
    Print ISSN: 0378-1119
    Electronic ISSN: 1879-0038
    Topics: Biology
    Published by Elsevier
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