Thermoprotective properties of small heat shock proteins from rice, tomato and Synechocystis sp. PCC6803 overexpressed in, and isolated from, Escherichia coli

2001 ◽  
Vol 28 (12) ◽  
pp. 1219
Author(s):  
Carl S. Pike ◽  
Joanne Grieve ◽  
Murray R. Badger ◽  
G. Dean Price
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Photosystem Ii ◽  
Heat Shock Proteins ◽  
Thermal Aggregation ◽  
E Coli ◽  
Synechococcus Sp ◽  
Shock Proteins ◽  
Synechocystis Sp

The present study forms part of a program investigating the role of small heat shock proteins (sHSPs) in the acquired and transgenic thermotolerance of the cyanobacterium Synechococcus PCC7942. The genes for three minimally related sHSPs, OsHSP from Oryza sativacytoplasm, tom111 from Lycopersicon esculentumchloroplasts, and 6803 HSP from Synechocystis sp. PCC6803, were cloned into the Escherichia coli vector pTrcHisA, so as to produce an N-terminal polyhistidine tag. The genes were transformed into E. coli and overexpressed. The tagged HSPs were purified (not completely in the case of tom111) by immobilised metal affinity chromatography. The native proteins exhibited a high degree of oligomerisation when analysed by size-exclusion chromatography. All three proteins were able to protect malate dehydrogenase (MDH) from in vitro thermal aggregation. They could also protect several soluble proteins in E. coli extracts from thermal aggregation in vitro, as well as protecting phycocyanin in extracts from Synechococcus sp. PCC7942. None of the proteins were able to protect photosystem II (measured as ΦPSII, the effective quantum fluorescence yield of PSII) of thylakoids isolated from Synechococcus sp. PCC7942 from heat damage in vitro, although in vivo, after acclimation, photosystem II did exhibit acquired thermotolerance.

In Vitro Holdase Activity of E. coli Small Heat-Shock Proteins IbpA, IbpB and IbpAB: A Biophysical Study with Some Unconventional Techniques

2014 ◽  
Vol 21 (6) ◽  
pp. 564-571 ◽  
Author(s):  
Sourav Roy ◽  
Monobesh Patra ◽  
Suman Nandy ◽  
Milon Banik ◽  
Rakhi Dasgupta ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Small Heat Shock Proteins ◽  
E Coli ◽  
Biophysical Study ◽  
Shock Proteins

scholarly journals Functional Analysis of Genes Comprising the Locus of Heat Resistance in Escherichia coli

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Ryan Mercer ◽  
Oanh Nguyen ◽  
Qixing Ou ◽  
Lynn McMullen ◽  
Michael G. Gänzle
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Heat Resistance ◽  
Growth Phase ◽  
Genomic Island ◽  
Enteric Bacteria ◽  
Content Type ◽  
E Coli ◽  
Shock Proteins

ABSTRACT The locus of heat resistance (LHR) is a 15- to 19-kb genomic island conferring exceptional heat resistance to organisms in the family Enterobacteriaceae, including pathogenic strains of Salmonella enterica and Escherichia coli. The complement of LHR-comprising genes that is necessary for heat resistance and the stress-induced or growth-phase-induced expression of LHR-comprising genes are unknown. This study determined the contribution of the seven LHR-comprising genes yfdX1 GI, yfdX2, hdeD GI, orf11, trx GI, kefB, and psiE GI by comparing the heat resistances of E. coli strains harboring plasmid-encoded derivatives of the different LHRs in these genes. (Genes carry a subscript “GI” [genomic island] if an ortholog of the same gene is present in genomes of E. coli.) LHR-encoded heat shock proteins sHSP20, ClpKGI, and sHSPGI are not sufficient for the heat resistance phenotype; YfdX1, YfdX2, and HdeD are necessary to complement the LHR heat shock proteins and to impart a high level of resistance. Deletion of trx GI, kefB, and psiE GI from plasmid-encoded copies of the LHR did not significantly affect heat resistance. The effect of the growth phase and the NaCl concentration on expression from the putative LHR promoter p2 was determined by quantitative reverse transcription-PCR and by a plasmid-encoded p2:GFP promoter fusion. The expression levels of exponential- and stationary-phase E. coli cells were not significantly different, but the addition of 1% NaCl significantly increased LHR expression. Remarkably, LHR expression in E. coli was dependent on a chromosomal copy of evgA. In conclusion, this study improved our understanding of the genes required for exceptional heat resistance in E. coli and factors that increase their expression in food. IMPORTANCE The locus of heat resistance (LHR) is a genomic island conferring exceptional heat resistance to several foodborne pathogens. The exceptional level of heat resistance provided by the LHR questions the control of pathogens by current food processing and preparation techniques. The function of LHR-comprising genes and their regulation, however, remain largely unknown. This study defines a core complement of LHR-encoded proteins that are necessary for heat resistance and demonstrates that regulation of the LHR in E. coli requires a chromosomal copy of the gene encoding EvgA. This study provides insight into the function of a transmissible genomic island that allows otherwise heat-sensitive enteric bacteria, including pathogens, to lead a thermoduric lifestyle and thus contributes to the detection and control of heat-resistant enteric bacteria in food.

Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP

1988 ◽  
Vol 8 (3) ◽  
pp. 1206-1215
Author(s):  
C F Clarke ◽  
K Cheng ◽  
A B Frey ◽  
R Stein ◽  
P W Hinds ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Protein Complexes ◽  
P53 Protein ◽  
Immunoaffinity Column ◽  
E Coli ◽  
Shock Proteins

Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.

The Escherichia coli chaperones involved in DNA replicadon

1993 ◽  
Vol 339 (1289) ◽  
pp. 271-278 ◽  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Conformational Changes ◽  
Heat Shock Genes ◽  
Dnab Helicase ◽  
Initiation Of Dna Replication ◽  
Shock Proteins

Mutadons in the Escherichia coli heat shock genes, dnaK , dnaJ or grpE , alter host DNA and RNA synthesis, degradation of other proteins, cell division and expression of other heat shock genes. They also block the initiation of DNA replication of bacteriophages λ and P1, and the mini-F plasmid. An in vitro λDNA replication system, composed entirely of purified components, enabled us to describe the molecular mechanism of the dnaK , dnaJ and grpE gene products. DnaK , the bacterial hsp 70 homologue, releases λP protein from the preprimosomal complex in an ATP- and DnaJ-dependent reaction (GrpEindependent initiation of λDNA replication). In this paper, I show that, when GrpE is present, λP protein is not released from the preprimosomal complex, rather it is translocated within the complex in such a way that it does not inhibit DnaB helicase activity. Translocation of λP triggers the initiation event allowing DnaB helicase to unwind DNA near the ori λ sequence, leading to efficient λDNA replication. Chaperone activity of the DnaK -DnaJ-GrpE system is first manifested in the selective binding of these heat shock proteins to the preprimosomal complex, followed by its ATP-dependent rearrangement. I show that DnaJ not only tags the preprimosomal complex for recognition by DnaK, but also stabilizes the multi-protein structure. GrpE also participates in the binding of DnaK to the preprimosomal complex by increasing DnaK ’s affinity to those λP proteins which are already associated with DnaJ. After attracting DnaK to the preprimosomal complex, DnaJ and GrpE stimulate the ATPase activity of DnaK , triggering conformational changes in DnaK which are responsible for the rearrangement of proteins in the preprimosomal complex and recycling of these heat shock proteins. The role of DnaK , DnaJ and GrpE in λDNA replication is in sharp contrast to our understanding of their role in the oriC , P1, and probably mini-F DNA replication systems. In the cases of oriC and P1 DNA replication, these heat shock proteins activate initiation factors before they are in contact with DNA, and are not required during the subsequent steps leading to the initiation of DNA replication. The common feature of DnaK , DnaJ and GrpE action in these systems is their ATP-dependent disaggregation or rearrangement of protein complexes formed before or during initiation of DNA replication.

scholarly journals Heat Shock Protein-Mediated Resistance to High Hydrostatic Pressure in Escherichia coli

2004 ◽  
Vol 70 (5) ◽  
pp. 2660-2666 ◽  
Author(s):  
Abram Aertsen ◽  
Kristof Vanoirbeek ◽  
Philipp De Spiegeleer ◽  
Jan Sermon ◽  
Kristel Hauben ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
High Pressure ◽  
Hydrostatic Pressure ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
High Hydrostatic Pressure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
E Coli ◽  
Shock Proteins

ABSTRACT A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i) the expression of rpoH, encoding the heat shock-specific sigma factor σ32, was also induced by high pressure; (ii) heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.

scholarly journals Multiple layers of control govern expression of the Escherichia coli ibpAB heat-shock operon

Microbiology ◽  
2011 ◽  
Vol 157 (1) ◽  
pp. 66-76 ◽  
Author(s):  
Lena C. Gaubig ◽  
Torsten Waldminghaus ◽  
Franz Narberhaus
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Transcriptional Control ◽  
Rnase E ◽  
Translation Control ◽  
E Coli ◽  
Complex Process ◽  
Shock Proteins

The Escherichia coli ibpAB operon encodes two small heat-shock proteins, the inclusion-body-binding proteins IbpA and IbpB. Here, we report that expression of ibpAB is a complex process involving at least four different layers of control, namely transcriptional control, RNA processing, translation control and protein stability. As a typical member of the heat-shock regulon, transcription of the ibpAB operon is controlled by the alternative sigma factor σ 32 (RpoH). Heat-induced transcription of the bicistronic operon is followed by RNase E-mediated processing events, resulting in monocistronic ibpA and ibpB transcripts and short 3′-terminal ibpB fragments. Translation of ibpA is controlled by an RNA thermometer in its 5′ untranslated region, forming a secondary structure that blocks entry of the ribosome at low temperatures. A similar structure upstream of ibpB is functional in vitro but not in vivo, suggesting downregulation of ibpB expression in the presence of IbpA. The recently reported degradation of IbpA and IbpB by the Lon protease and differential regulation of IbpA and IbpB levels in E. coli are discussed.

scholarly journals TNF- , IL-1 , IL-6 and ICAM-1 Expression in Human Keratinocytes Stimulated in Vitro with Escherichia Coli Heat-Shock Proteins

Microbiology ◽  
1997 ◽  
Vol 143 (1) ◽  
pp. 45-53 ◽  
Author(s):  
A. Marcatili ◽  
G. C. de I'Ero ◽  
M. Galdiero ◽  
A. Folgore ◽  
G. Petrillo
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Human Keratinocytes ◽  
Shock Proteins
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