Dataset Information

The Escherichia coli heat shock gene htpY: mutational analysis, cloning, sequencing, and transcriptional regulation.

ABSTRACT: We have identified a new heat shock gene, designated htpY, located 700 bp upstream of the dnaK dnaJ operon. We cloned it and showed that it is transcribed clockwise vis-à-vis the Escherichia coli genetic map, in the same direction as the dnaK dnaJ operon. The htpY gene encodes a 21,193-Da polypeptide. Promoter mapping experiments and Northern (RNA) analysis showed that the htpY gene belongs to the classical heat shock gene family, because the transcription from its major promoter is under the positive control of the rpoH gene product (sigma 32) and resembles canonical E sigma 32-transcribed consensus promoter sequences. This conclusion has been strengthened by the construction and analysis of a phtpY-lacZ promoter fusion. Despite the fact that htpY null bacteria are viable, the expression of various E sigma 32 heat shock promoters is significantly decreased, suggesting that HtpY plays an important role in the regulation of the heat shock response. Consistent with this interpretation, overproduction of the HtpY protein results in a generalized increase of the heat shock response in E. coli.

SUBMITTER: Missiakas D

PROVIDER: S-EPMC204563 | biostudies-other | 1993 May

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

The Escherichia coli heat shock gene htpY: mutational analysis, cloning, sequencing, and transcriptional regulation.

Missiakas D D Georgopoulos C C Raina S S

Journal of bacteriology 19930501 9

We have identified a new heat shock gene, designated htpY, located 700 bp upstream of the dnaK dnaJ operon. We cloned it and showed that it is transcribed clockwise vis-à-vis the Escherichia coli genetic map, in the same direction as the dnaK dnaJ operon. The htpY gene encodes a 21,193-Da polypeptide. Promoter mapping experiments and Northern (RNA) analysis showed that the htpY gene belongs to the classical heat shock gene family, because the transcription from its major promoter is under the po ...[more]

PMID: 8478327

Similar Datasets

Project description:BackgroundHigh temperatures cause a suite of problems for cells, including protein unfolding and aggregation; increased membrane fluidity; and changes in DNA supercoiling, RNA stability, transcription and translation. Consequently, enhanced thermotolerance can evolve through an unknown number of genetic mechanisms even in the simple model bacterium Escherichia coli. To date, each E. coli study exploring this question resulted in a different set of mutations. To understand the changes that can arise when an organism evolves to grow at higher temperatures, we sequenced and analyzed two previously described E. coli strains, BM28 and BM28 ΔlysU, that have been laboratory adapted to the highest E. coli growth temperature reported to date.ResultsWe found three large deletions in the BM28 and BM28 ΔlysU strains of 123, 15 and 8.5 kb in length and an expansion of IS10 elements. We found that BM28 and BM28 ΔlysU have considerably different genomes, suggesting that the BM28 culture that gave rise to BM28 and BM28 ΔlysU was a mixed population of genetically different cells. Consistent with published findings of high GroESL expression in BM28, we found that BM28 inexplicitly carries the groESL bearing plasmid pOF39 that was maintained simply by high-temperature selection pressure. We identified over 200 smaller insertions, deletions, single nucleotide polymorphisms and other mutations, including changes in master regulators such as the RNA polymerase and the transcriptional termination factor Rho. Importantly, this genome analysis demonstrates that the commonly cited findings that LysU plays a crucial role in thermotolerance and that GroESL hyper-expression is brought about by chromosomal mutations are based on a previous misinterpretation of the genotype of BM28.ConclusionsThis whole-genome sequencing study describes genetically distinct mechanisms of thermotolerance evolution from those found in other heat-evolved E. coli strains. Studying adaptive laboratory evolution to heat in simple model organisms is important in the context of climate change. It is important to better understand genetic mechanisms of enhancing thermotolerance in bacteria and other organisms, both in terms of optimizing laboratory evolution methods for various organisms and in terms of potential genetic engineering of organisms most at risk or most important to our societies and ecosystems.

Project description:The 93 kDa ClpB (ClpB93) is a heat shock protein and has a protein-activated ATPase activity. To define the role of the two ATP-binding sites in ClpB93, site-directed mutagenesis was performed to replace Lys212 or Lys611 with Thr or Glu. All of the mutant proteins hydrolysed ATP at a higher rate than that seen with ClpB93 at ATP concentrations up to 2 mM. However, ClpB93 carrying mutations in both of the ATP-binding sites could not cleave ATP. Thus any of the two ATP-binding sites seems to be capable of supporting the ATPase activity of ClpB93. The ATPase activities of both ClpB93/K212T and ClpB93/K212E were gradually decreased when ATP concentrations were increased above 2 mM, unlike those of ClpB93, ClpB93/K611T and ClpB93/K611E, which showed a typical saturation curve. Furthermore ADP inhibited ATP hydrolysis by ClpB93/K212T and ClpB93/K212E more effectively than that by the latter proteins, suggesting that the mutations in the first ATP-binding site result in an increase in the affinity of ADP for the second site in ClpB93. In addition, all of the purified ClpB93 and its mutant forms behaved as an oligomer of 400-450 kDa on a Sephacryl S-300 gel-filtration column, whether or not ATP was present. Thus the binding of ATP to either of the two sites seems not to be essential for oligomerization of ClpB93. Although a low-copy plasmid carrying clpB93 could rescue the sensitivity of a clpB-null mutant cell at 52 degreesC, none of the plasmids carrying the mutations in the ATP-binding sites could. Furthermore, incubation at 52 degreesC resulted in a gradual loss of the ATPase activity of ClpB93 carrying the mutations in either of the two ATP-binding sites, but not of the parental ClpB93, indicating that the mutant proteins have a greater tendency to denature at this temperature than the parental ClpB93. These results suggest that both of the ATP-binding sites in ClpB have an important role in maintaining the thermotolerance of the protein and hence in the survival of Escherichia coli at high temperatures.

Dataset Information

The Escherichia coli heat shock gene htpY: mutational analysis, cloning, sequencing, and transcriptional regulation.

Publications

The Escherichia coli heat shock gene htpY: mutational analysis, cloning, sequencing, and transcriptional regulation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets