A segment of cold shock protein directs the folding of a combinatorial protein.
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It has been suggested that protein domains evolved by the non-homologous recombination of building blocks of subdomain size. In earlier work we attempted to recapitulate domain evolution in vitro. We took a polypeptide segment comprising three beta-strands in the monomeric, five-stranded beta-barrel cold shock protein (CspA) of Escherichia coli as a building block. This segment corresponds to a complete exon in homologous eukaryotic proteins and includes residues that nucleate folding in CspA. We recombined this segment at random with fragments of natural proteins and succeeded in generating a range of folded chimaeric proteins. We now present the crystal structure of one such combinatorial protein, 1b11, a 103-residue polypeptide that includes segments from CspA and the S1 domain of the 30S ribosomal subunit of E. coli. The structure reveals a segment-swapped, six-stranded beta-barrel of unique architecture that assembles to a tetramer. Surprisingly, the CspA segment retains its structural identity in 1b11, recapitulating its original fold and deforming the structure of the S1 segment as necessary to complete a barrel. Our work provides structural evidence that (i) random shuffling of nonhomologous polypeptide segments can lead to folded proteins and unique architectures, (ii) many structural features of the segments are retained, and (iii) some segments can act as templates around which the rest of the protein folds. (+info)
Gene expression regulation by the Curli activator CsgD protein: modulation of cellulose biosynthesis and control of negative determinants for microbial adhesion.
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Curli fibers, encoded by the csgBAC genes, promote biofilm formation in Escherichia coli and other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain of E. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from the csgBAC promoter; however, csgBAC expression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. The cspA and fecR genes, encoding regulatory proteins responding to cold shock and to iron, respectively, and yoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of the yoaD gene negatively affects cell aggregation, while yoaD inactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation of fecR results in significant increases in both cell aggregation and biofilm formation, while the effects of cspA are not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of the yoaD gene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of the fecR gene. (+info)
Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems.
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Although the cold-shock response has now been studied in a number of different organisms for several decades, it is only in the last few years that we have begun to understand the molecular mechanisms that govern adaptation to cold stress. Notably, all organisms from prokaryotes to plants and higher eukaryotes respond to cold shock in a comparatively similar manner. The general response of cells to cold stress is the elite and rapid overexpression of a small group of proteins, the so-called CSPs (cold-shock proteins). The most well characterized CSP is CspA, the major CSP expressed in Escherichia coli upon temperature downshift. More recently, a number of reports have shown that exposing yeast or mammalian cells to sub-physiological temperatures (<30 or <37 degrees C respectively) invokes a co-ordinated cellular response involving modulation of transcription, translation, metabolism, the cell cycle and the cell cytoskeleton. In the present review, we summarize the regulation and role of cold-shock genes and proteins in the adaptive response upon decreased temperature with particular reference to yeast and in vitro cultured mammalian cells. Finally, we present an integrated model for the co-ordinated responses required to maintain the viability and integrity of mammalian cells upon mild hypothermic cold shock. (+info)
Global effects of homocysteine on transcription in Escherichia coli: induction of the gene for the major cold-shock protein, CspA.
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Homocysteine (Hcy) is a thiol-containing amino acid that is considered to be medically important because it is linked to the development of several life-threatening diseases in humans, including cardiovascular disease and stroke. It inhibits the growth of Escherichia coli when supplied in the growth medium. Growth inhibition is believed to arise as a result of partial starvation for isoleucine, which occurs because Hcy perturbs the biosynthesis of this amino acid. This study attempted to further elucidate the inhibitory mode of action of Hcy by examining the impact of exogenously supplied Hcy on the transcriptome. Using gene macroarrays the transcript levels corresponding to 68 genes were found to be reproducibly altered in the presence of 0.5 mM Hcy. Of these genes, the biggest functional groups affected were those involved in translation (25 genes) and in amino acid metabolism (19 genes). Genes involved in protection against oxidative stress were repressed in Hcy-treated cells and this correlated with a decrease in catalase activity. The gene showing the strongest induction by Hcy was cspA, which encodes the major cold-shock protein CspA. RT-PCR and reporter fusion experiments confirmed that cspA was induced by Hcy. Induction of cspA by Hcy was not caused by nutritional upshift, a stimulus known to induce CspA expression, nor was it dependent on the presence of a functional CspA protein. The induction of cspA by Hcy was suppressed when isoleucine was included in the growth medium. These data suggest that the induction of CspA expression in the presence of Hcy occurs because of a limitation for isoleucine. The possibility that Hcy-induced cspA expression is triggered by translational stalling that occurs when the cells are limited for isoleucine is discussed. (+info)
Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli.
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Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process. (+info)
Bacterial toxicity of potassium tellurite: unveiling an ancient enigma.
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Biochemical, genetic, enzymatic and molecular approaches were used to demonstrate, for the first time, that tellurite (TeO(3) (2-)) toxicity in E. coli involves superoxide formation. This radical is derived, at least in part, from enzymatic TeO(3) (2-) reduction. This conclusion is supported by the following observations made in K(2)TeO(3)-treated E. coli BW25113: i) induction of the ibpA gene encoding for the small heat shock protein IbpA, which has been associated with resistance to superoxide, ii) increase of cytoplasmic reactive oxygen species (ROS) as determined with ROS-specific probe 2'7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), iii) increase of carbonyl content in cellular proteins, iv) increase in the generation of thiobarbituric acid-reactive substances (TBARs), v) inactivation of oxidative stress-sensitive [Fe-S] enzymes such as aconitase, vi) increase of superoxide dismutase (SOD) activity, vii) increase of sodA, sodB and soxS mRNA transcription, and viii) generation of superoxide radical during in vitro enzymatic reduction of potassium tellurite. (+info)
Role of RNA structure and susceptibility to RNase E in regulation of a cold shock mRNA, cspA mRNA.
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Degradation of the cspA mRNA in vivo is very rapid at temperatures greater than 30 degrees C and is moderately dependent on RNase E. Investigations in vitro show that degradosomes prepared from normal or cold-shocked cultures cleave the cspA mRNA preferentially at a single site in vitro between two stem-loops approximately 24 residues 3' to the termination codon and approximately 31 residues from the 3' end. The site of cleavage is independent of the temperature and largely independent of the phosphorylation status of the 5' end of cspA mRNA. A 5' stem-loop, potential occlusion of the initiation and termination codons, temperature-dependent translational efficiency, and the position of the RNase E cleavage site can explain the differential stability of the cspA mRNA. (+info)
Dimorphic aggregation behavior of a fusion polypeptide incorporating a stable protein domain (EGFP) with an amyloidogenic sequence (retroCspA).
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