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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The membrane-anchored DjlA protein represents the third member of the DnaJ ‘J-domain’ family of Escherichia coli that includes DnaJ and CbpA. DjlA possesses a J-domain at its extreme C-terminus but shares no additional homology with DnaJ. Our genetic analysis suggests that DjlA acts in concert with the RcsB/C two-component signal transduction system to augment induction of the cps (capsular polysaccharide) operon and synthesis of colanic acid mucoid capsule. The DjlA J-domain is essential for the observed stimulation of this pathway as deletion, or introduction of the mutation H233Q, within the highly conserved HPD tripeptide abolished all inducing activity. Deletion of the transmembrane anchor sequence also abolished all inducing activity. djlA is not an essential gene under all conditions tested, nor is it essential for mucoid capsule biosynthesis; however, strong overexpression leads to rapid loss of cell viability suggesting that the gene is normally tightly regulated. Northern analysis revealed that djlA message was extremely unstable but could be induced or stabilized in response to cold shock. The activation of the cps operon by DjlA is dependent upon both DnaK(Hsp70) and GrpE, and therefore we propose a role for DjlA, together with this chaperone machine, as a novel regulator of a two-component histidine kinase signal transduction pathway.
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  • 2
    Electronic Resource
    Electronic Resource
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Genetics 34 (2000), S. 439-456 
    ISSN: 0066-4197
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Biology
    Notes: Abstract Early genetic studies identified the Escherichia coli groES and groEL genes because mutations in them blocked the growth of bacteriophages lamba and T4. Subsequent genetic and biochemical analyses have shown that GroES and GroEL constitute a chaperonin machine, absolutely essential for E. coli growth, because it is needed for the correct folding of many of its proteins. In spite of very little sequence identity to GroES, the bacteriophage T4-encoded Gp31 protein and the bacteriophage RB49-encoded CocO protein are bona fide GroEL cochaperonins, even capable of substituting for GroES in E. coli growth. A major functional distinction is that only Gp31 and CocO can assist GroEL in the correct folding of Gp23, the major bacteriophage capsid protein. Conserved structural features between CocO and Gp31, which are absent from GroES, highlight their potential importance in specific cochaperonin function.
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology reviews 17 (1995), S. 0 
    ISSN: 1574-6976
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract: The Escherichia coli/bacteriophage λ genetic interaction system has been used to uncover the existence of various biological machines. The starting point of all these studies was the isolation and characterization of E. coli mutants that blocked λ growth, and the corresponding λ compensatory mutations. In this manner, the λN-promoted transcriptional anti-termination machine was discovered composed of the NusA/NusB/NusE/NusG host proteins. In addition, the DnaK and GroEL chaperone machines were discovered composed of DnaK/DnaJ/GrpE and GroES/GroEL heat shock proteins. The individual members of the DnaK and GroEL chaperone machines have been conserved throughout evolution in both function and structure. Their biological roles include a direct involvement in λ DNA replication and morphogenesis, the protection of proteins from aggregation, the disaggregation of various protein aggregates, the manipulation of protein structure and function, as well as the autoregulation of the heat shock response. The evolution of λ to extensively rely on the status of the heat shock response of E. coli is likely linked to its lytic versus lysogenic choice of lifestyle. The bacteriophage T4 gp31 protein has been purified and shown to substitute for many of GroES' co-chaperonin activities.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 20 (1996), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The htrB gene was discovered because its insertional inactivation interfered with Escherichia coli growth and viability at temperatures above 32.5°C, as a result of accumulation of phospholipids. The msbA gene was originally discovered because when cloned on a low-copy-number plasmid vector it was able to suppress the temperature-sensitive growth phenotype of an htrB null mutant as well as the accumulation of phospholipids. The msbA gene product belongs to the superfamily of ABC transporters, a universally conserved family of proteins characterized by a highly conserved ATP-binding domain. The msbA gene is essential for bacterial viability at all temperatures. In order to understand the physiological role of the MsbA protein, we mutated the ATP-binding domain using random PCR mutagenesis. Six independent mutants were isolated and characterized. Four of these mutations resulted in single-amino-acid substitutions in non-conserved residues and were able to support cell growth at 30°C but not at 43°C. The remaining two mutations behaved as recessive lethals, and resulted in single-amino-acid substitutions in Walker motif B, one of the two highly conserved regions of the ATP-binding domain. Despite the fact that neither of these two mutant proteins can support E. coli growth, they both retained the ability to bind ATP in vitro. In addition, we present evidence to show that W-acetyl [3H]-glucosamine, a precursor of lipopolysaccharides, accumulates at the non-permissive temperature in the inner membrane of either htrB null or msbA conditional lethal strains. Translocation of the precursor to the outer membrane is restored by transformation with a plasmid containing the wild-type msbA gene. A possible role for MsbA
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We report the characterization of the msbA gene, isolated as a multicopy suppressor of the HtrB temperature-sensitive phenotype. The msbA gene maps to 20.5 min on the Escherichia coli genetic map and encodes a protein with an estimated molecular mass of 64460 Da, with the properties of an integral membrane protein. The amino acid sequence of MsbA is very similar to those of the family of ATP-dependent translocators, which includes the haemolysin B protein of E. coli and the mammalian multidrug resistance (MDR) proteins. Mutational analysis of msbA indicates that it may form an operon with a downstream gene, orfE, and that both of these genes are essential for bacterial viability under all growth conditions tested.
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  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The Escherichia coliσ32 transcriptional regulator has been shown to be degraded both in vivo and in vitro by the FtsH (HflB) protease, a member of the AAA protein family. In our attempts to study this process in detail, we found that two σ32 mutants lacking 15–20 C-terminal amino acids had substantially increased half-lives in vivo or in vitro, compared with wild-type σ32. A truncated version of σ32, σ32CΔ, was purified to homogeneity and shown to be resistant to FtsH-dependent degradation in vitro, suggesting that FtsH initiates σ32 degradation from its extreme C-terminal region. Purified σ32CΔ interacted with the DnaK and DnaJ chaperone proteins in a fashion similar to that of wild-type σ32. However, in contrast to wild-type σ32, σ32CΔ was largely deficient in its in vivo and in vitro interaction with core RNA polymerase. As a consequence, the truncated σ32 protein was completely non-functional in vivo, even when overproduced. Furthermore, it is shown that wild-type σ32 is protected from degradation by FtsH when complexed to the RNA polymerase core, but sensitive to proteolysis when in complex with the DnaK chaperone machine. Our results are in agreement with the proposal that the capacity of the DnaK chaperone machine to autoregulate its own synthesis negatively is simply the result of its ability to sequester σ32 from RNA polymerase, thus making it accessible to degradation by the FtsH protease.
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford BSL : Blackwell Science Ltd, UK
    Molecular microbiology 30 (1998), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: DnaJ is a universally conserved heat shock protein involved in protein folding. DnaJ contains four conserved domains. The N-terminal ‘J-domain’ has been shown to be responsible for the recruitment of its specific DnaK partner protein. The ‘Gly/Phe’- and ‘Cys-rich’ domains have been implicated in stabilizing interactions with DnaK. DnaJ is also able to interact independently with unfolded or native polypeptides. Very little is known regarding such binding/chaperone abilities, but it has been suggested that the least conserved carboxy-terminal domain could contribute to these properties. To gain insight into the biological activity of this fourth domain, we deleted two relatively conserved patches of amino acid residues, a ‘G-rich’ cluster and a ‘G–D–L–Y–V’ motif, resulting in the DnaJΔ[230–238] and DnaJΔ[242–246] mutant proteins respectively. Both mutant proteins are partially defective in stimulating the ATPase activity of DnaK and in preventing aggregation of firefly luciferase in vitro. Both mutants have lost the ability to regulate the σ32-dependent heat shock response, as shown in vivo using a heat shock transcriptional fusion. Furthermore, and unlike wild-type DnaJ, DnaJΔ[242–246] is unable to assist the DnaK-dependent refolding of denatured luciferase. In agreement with these results, we found that DnaJΔ[242–246] is unable to restore either the temperature-sensitive phenotype or the motility defect of a dnaJ null mutation. Substitution of amino acids [242–246] by five alanines leads to similar phenotypic defects, suggesting that altering the ‘G–D–L–Y–V’ motif leads to partial loss of DnaJ activity. Our data clearly support a role in the intrinsic chaperone/substrate binding ability of the carboxy-terminal domain of DnaJ.
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  • 8
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The σE (RpoE) transcription factor of Escherichia coli regulates the expression of genes whose products are devoted to extracytoplasmic activities. The σE regulon is induced upon misfolding of proteins in the periplasm or the outer membrane. Similar to other alternative sigma factors, the activity of σE is tightly regulated in E. coli. We have previously shown that σE is positively autoregulated at the transcriptional level. DNA sequencing, coupled with transcriptional analyses, have shown that σE is encoded by the first gene of a four-gene operon. The second gene of this operon, rseA, encodes an anti-σE activity. This was demonstrated at both the genetic and biochemical levels. For example, mutations in rseA constitutively increase σE activity. Consistent with this, overproduction of RseA leads to an inhibitory effect on σE activity. Topological analysis of RseA suggests the existence of one transmembrane domain, with the N-terminal part localized in the cytoplasm. Overproduction of this N-terminal domain alone was shown to inhibit σE activity. These observations were confirmed in vitro, because either purified RseA or only its purified N-terminal domain inhibited transcription from EσE-dependent promoters. Furthermore, RseA and σE co-purify, and can be co-immunoprecipitated, and chemically cross-linked. The σE activity is further modulated by the products of the remaining genes in this operon, rseB and rseC. RseB is a periplasmic protein, which negatively regulates σE activity and specifically interacts with the C-terminal periplasmic domain of RseA. In contrast, RseC is an inner membrane protein that positively modulates σE activity. Most of these protein–protein interactions were verified in vivo using the yeast two-hybrid system.
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  • 9
    ISSN: 1072-8368
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Two models are being considered for the mechanism of chaperonin-assisted protein folding in E. coli: (i) GroEL/GroES act primarily by enclosing substrate polypeptide in a folding cage in which aggregation is prevented during folding. (ii) GroEL mediates the repetitive unfolding of misfolded ...
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  • 10
    ISSN: 1072-8368
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Medicine
    Notes: [Auszug] Nature Struct. Biol. 5, 977– 985 (1998). The text of Table 1 contained several errors, which we regret. The correct version is printed ...
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