Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis

Schmidt, Alexander; Schiesswohl, Michael; Völker, Uwe; Hecker, Michael; Schumann, Wolfgang

doi:10.1128/jb.174.12.3993-3999.1992

Cited by 122 publications

(100 citation statements)

References 36 publications

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“…The more denatured proteins are removed from the cell, the more GroE chaperonins are available to convert inactive HrcA into its active form (12). This in turn leads to the shut off of the Class I heat shock genes under constantly high temperature conditions (7)(8)(9). This direct HrcA-GroE interaction model has been challenged by Minder et al (29) whom did not see a more efficient retardation of target DNA by HrcA of Bradyrhizobium japonicum in the presence of added GroEL.…”

Section: Discussionmentioning

confidence: 94%

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Isolation and Analysis of Mutant Alleles of the Bacillus subtilis HrcA Repressor with Reduced Dependency on GroE Function

Reischl

Wiegert

Schumann

2002

Journal of Biological Chemistry

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The hrcA gene of Bacillus subtilis codes for a transcriptional repressor protein that negatively regulates expression of the heptacistronic dnaK and the bicistronic groE operon by binding to an operator-element called CIRCE. Recently, we have published data suggesting that the activity of HrcA is modulated by the GroE chaperonin system. Biochemical analyses of the HrcA protein have been hampered so far by its strong tendency to aggregate. Here, a genetic method was used to isolate mutant forms of HrcA with increased activity under conditions of decreased GroE function. One of these mutant forms (HrcA114) containing five amino acid replacements exhibited enhanced solubility when overexpressed. HrcA114 purified under native conditions produced two retarded CIRCE-containing DNA fragments in band shift experiments. The amount of the larger fragment increased after addition of GroEL, GroES, and ATP but decreased when ATP was replaced by the nonhydrolyzable ATP analog ATP␥S. DNase I footprinting experiments exhibited full protection of the CIRCE element and neighboring nucleotides in an asymmetric way. An in vitro binding assay using affinity chromatography showed direct and specific interaction between HrcA114 and GroEL. All these experimental data are in full agreement with our previously published model that HrcA needs the GroE chaperonin system for activation.Bacteria encode genetic systems allowing them to adapt to many stressful situations, including high and low temperature, hyperosmotic and oxidative stress, and severe DNA damage (1). The best-studied stress response is the so-called heat shock response, which is induced after a sudden increase in temperature. This response is characterized by the transiently enhanced synthesis of a group of proteins collectively known as heat shock proteins encoded by heat shock genes. Work carried out over the last 5 years has revealed that in most eubacteria heat shock genes are organized in two and more regulons, where each regulon is either under positive control of an alternative sigma factor or under negative control of a transcriptional repressor (2-5).In Bacillus subtilis, three different regulons have been identified so far, where Class I heat shock genes are under the negative control of the HrcA transcriptional repressor. This protein binds to an operator designated CIRCE 1 (Controlling Inverted Repeat of Chaperone Expression) (6), which precedes the heptacistronic dnaK and the bicistronic groE operon (7-9). Upon a heat shock, HrcA dissociates from its operators leading to a transient induction of the two operons followed by rebinding after about 10 min (7,8,10). The pertinent question concerning all heat shock regulators is how the activity of these proteins is modulated after a heat shock. In the present case, we have presented data suggesting that the activity of HrcA is modulated by the GroE chaperonin system (11). HrcA is maintained in an active conformation able to bind to CIRCE through GroE. Under conditions of increased formation of nonnative proteins in th...

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Section: Discussionmentioning

confidence: 94%

“…This protein binds to an operator designated CIRCE 1 (Controlling Inverted Repeat of Chaperone Expression) (6), which precedes the heptacistronic dnaK and the bicistronic groE operon (7)(8)(9). Upon a heat shock, HrcA dissociates from its operators leading to a transient induction of the two operons followed by rebinding after about 10 min (7,8,10).…”

mentioning

confidence: 99%

Isolation and Analysis of Mutant Alleles of the Bacillus subtilis HrcA Repressor with Reduced Dependency on GroE Function

Reischl

Wiegert

Schumann

2002

Journal of Biological Chemistry

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“…Experiments are in progress to determine if these three genes encode heat-shock proteins, and if they are involved in the regulation of the heat-shock response of class I genes. The groE operon of B. subtilis is bicistronic and consists of the genes groES and groEL (Schmidt et al, 1992;Li and Wong, 1992). This genomic organization has been described in many other bacterial species studied to date for the groE operon.…”

Section: Class I Heat-shock Genes Are Probably Regulated By a Repressormentioning

confidence: 96%

“…The dnaK operon consists of at least four genes (orf39-grpE-dnaK-dnaJ ), the latter three of which exhibit significant homology to genes known from E. coli and other eubacterial species (Wetzstein et al, 1992;Schmidt et al, 1992). In contrast to E. coli, the genes grpE, dnaK, and dnaJ, the products of which constitute the DnaK chaperone machine (Georgopoulos and Welch, 1993), are within one operon.…”

Section: Class I Heat-shock Genes Are Probably Regulated By a Repressormentioning

confidence: 99%

Heat‐shock and general stress response in Bacillus subtilis

Hecker

Schumann

Völker

1996

Molecular Microbiology

563

532

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SummaryThe 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

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“…subtilis (56) and the dnaK and groESL operons of C. acetobutylicum (5,43). A data base search for additional genes preceded by this inverted repeat led to the discovery of some more examples, which are listed in Table 2.…”

Section: Discussionmentioning

confidence: 99%

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis

et al. 1992

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By using an internal part of the dnaK gene from BaciUlus megaterium as a probe, a 5.2-kb Hindlll fragment of chromosomal DNA of BaciUlus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf9-grpEdnaK-dnaJ. orJ39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orJ39 (Si and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in a-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orJ39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 2230 on the B. subtilis genetic map.The heat shock response is a homeostatic mechanism that enables cells to survive a variety of environmental stresses. It is characterized by the increased synthesis of a group of evolutionarily conserved proteins, heat shock proteins (HSPs), and is a universal feature of both prokaryotic and eukaryotic cells (35). When Eschertichia coli cells are shifted to a high temperature, the synthesis of a set of about 20 HSPs transiently increases and then declines rapidly to steady-state levels characteristic of the new ambient temperature (44).The highly conserved HSPs perform similar functions in all organisms. One of these functions is the well-established regulation of protein-protein interactions by the chaperonins (69). One of the most abundant HSPs, HSP70, is highly conserved in evolution. It is found in such diverse organisms as E. coli, Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens (14, 35).The dnaK gene of E. coli was originally discovered because mutations in it blocked bacteriophage lambda DNA replication at all temperatures (22,23). Subsequently, the dnaK gene product was shown to be essential for E. coli viability at high and low temperatures (22,30,46,51,52), and genetic evid...

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Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis

Cited by 122 publications

References 36 publications

Isolation and Analysis of Mutant Alleles of the Bacillus subtilis HrcA Repressor with Reduced Dependency on GroE Function

Isolation and Analysis of Mutant Alleles of the Bacillus subtilis HrcA Repressor with Reduced Dependency on GroE Function

Heat‐shock and general stress response in Bacillus subtilis

Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis

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