groESL Expression Research Articles

Whole-genome sequencing analysis of two heat-evolved Escherichia coli strains

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.

Enhanced butyric acid tolerance and production by Class I heat shock protein-overproducing Clostridium tyrobutyricum ATCC 25755.

The response of Clostridium tyrobutyricum to butyric acid stress involves various stress-related genes, and therefore overexpression of stress-related genes can improve butyric acid tolerance and yield. Class I heat shock proteins (HSPs) play an important role in the process of protecting bacteria from sudden changes of extracellular stress by assisting protein folding correctly. The results of quantitative real-time PCR indicated that the Class I HSGs grpE, dnaK, dnaJ, groEL, groES, and htpG were significantly upregulated under butyric acid stress, especially the dnaK and groE operons. Overexpression of groESL and htpG could significantly improve the tolerance of C. tyrobutyricum to butyric acid, while overexpression of dnaK and dnaJ showed negative effects on butyric acid tolerance. Acid production was also significantly promoted by increased GroESL expression levels; the final butyric acid and acetic acid concentrations were 28.2 and 38% higher for C. tyrobutyricum ATCC 25755/groESL than for the wild-type strain. In addition, when fed-batch fermentation was carried out using cell immobilization in a fibrous-bed bioreactor, the butyric acid yield produced by C. tyrobutyricum ATCC 25755/groESL reached 52.2g/L, much higher than that for the control. The improved butyric acid yield is probably attributable to the high GroES and GroEL levels, which can stabilize the biosynthetic machinery of C. tyrobutyricum under extracellular butyric acid stress.

Most heat-tolerant rhizobia show high induction of major chaperone genes upon stress

The aims of this study were to evaluate the temperature stress tolerance of chickpea rhizobia and to investigate whether tolerance is related to the species or the site of origin of the isolates. Additionally, the molecular bases of temperature stress tolerance in rhizobia were investigated, by comparing the expression of chaperone genes dnaKJ and groESL in thermotolerant and thermosensitive isolates. Tolerance to cold, heat and heat shock was evaluated for 53 mesorhizobia obtained from several provinces of Portugal and assigned to different species. Associations between isolates' tolerance phenotype and several provinces of origin were found. Some species groups were found to differ significantly in their ability to tolerate temperature stress. Analysis of the dnaK and groESL expression by Northern hybridization, using isolates from three species groups, showed an increase in the transcripts levels with heat, but not with cold stress. Interestingly, a higher induction of chaperone genes was detected in heat-tolerant isolates when compared with that of sensitive isolates of the same species. To our knowledge, this is the first analysis of chaperone genes' expression comparing tolerant and sensitive strains. The present study suggests a relationship between higher transcriptional induction of major chaperone genes and higher tolerance to heat in rhizobia.

Differential regulation of groESL operon expression in response to heat and light in Anabaena

The HrcA protein is known to bind the cis-element CIRCE and repress expression of hsp60 in certain bacteria. However, recent data from cyanobacteria have seriously questioned the HrcA/CIRCE interaction paradigm. A hrcA null mutant showed constitutive expression of Hsp60 proteins [GroEL/Cpn60(GroEL2)], and an unexpected further increase in GroEL during temperature upshift, suggesting involvement of regulatory mechanisms other than HrcA in groESL expression in Anabaena. The negative regulation of both hsp60 genes [groEL and cpn60 (groEL2)] at CIRCE element was established by: (1) constitutive expression of Green Fluorescent Protein gene, tagged to Anabaena hsp60 promoters, in E. coli, and its repression upon co-expression of Anabaena HrcA and (2) specific binding of Anabaena HrcA to the CIRCE element. Deletion analysis of other cis-elements further distinguished (a) a photo-regulation by the K-box and (b) thermoregulation from a novel H-box, over and above the negative regulation by HrcA at CIRCE.

Functional and structural analysis of HrcA repressor protein from Caulobacter crescentus.

A large number of bacteria regulate chaperone gene expression during heat shock by the HrcA-CIRCE system, in which the DNA element called CIRCE serves as binding site for the repressor protein HrcA under nonstress conditions. In Caulobacter crescentus, the groESL operon presents a dual type of control. Heat shock induction is controlled by a sigma32-dependent promoter and the HrcA-CIRCE system plays a role in regulation of groESL expression under physiological temperatures. To study the activity of HrcA in vitro, we purified a histidine-tagged version of the protein, and specific binding to the CIRCE element was obtained by gel shift assays. The amount of retarded DNA increased significantly in the presence of GroES/GroEL, suggesting that the GroE chaperonin machine modulates HrcA activity. Further evidence of this modulation was obtained using lacZ transcription fusions with the groESL regulatory region in C. crescentus cells, producing different amounts of GroES/GroEL. In addition, we identified the putative DNA-binding domain of HrcA through extensive protein sequence comparison and constructed various HrcA mutant proteins containing single amino acid substitutions in or near this region. In vitro and in vivo experiments with these mutated proteins indicated several amino acids important for repressor activity.

HrcA is a negative regulator of the dnaK and groESL operons of Streptococcus pyogenes

The genome of Streptococcus pyogenes, an important human pathogen, encodes homologs of the principal bacterial heat shock proteins DnaK and GroES, -EL, as well as HrcA, a negative regulator of dnaK and groESL expression in other Gram-positive bacteria. Using nuclease protection assays to measure dnaK/groESL mRNA abundance and a “non-polar” insertion to disrupt hrcA, we demonstrate that heat shock triggers a 4- to 8-fold increase in dnaK and groESL-specific mRNAs within 5 min of the temperature shift and that HrcA is a negative regulator of S. pyogenes dnaK/groESL mRNA abundance in unstressed S. pyogenes. Although the loss of HrcA elevated dnaK and groESL mRNA levels under non-heat shock conditions, the relative abundance of these RNAs increased further in heat shocked S. pyogenes, suggesting an additional element contributing to their synthesis or stability.

The CIRCE element and its putative repressor control cell cycle expression of the Caulobacter crescentus groESL operon.

The groESL operon is under complex regulation in Caulobacter crescentus. In addition to strong induction after exposure to heat shock, under physiological growth conditions, its expression is subject to cell cycle control. Transcription and translation of the groE genes occur primarily in predivisional cells, with very low levels of expression in stalked cells. The regulatory region of groESL contains both a sigma32-like promoter and a CIRCE element. Overexpression of C. crescentus sigma32 gives rise to higher levels of GroEL and increased levels of the groESL transcript coming from the sigma32-like promoter. Site-directed mutagenesis in CIRCE has indicated a negative role for this cis-acting element in the expression of groESL only at normal growth temperatures, with a minor effect on heat shock induction. Furthermore, groESL-lacZ transcription fusions carrying mutations in CIRCE are no longer cell cycle regulated. Analysis of an hrcA null strain, carrying a disruption in the gene encoding the putative repressor that binds to the CIRCE element, shows constitutive synthesis of GroEL throughout the Caulobacter cell cycle. These results indicate a negative role for the hrcA gene product and the CIRCE element in the temporal control of the groESL operon.

Expression of the groESL operon is cell-cycle controlled in Caulobacter crescentus.

The Caulobacter crescentus groESL operon was cloned, sequenced and found to be homologous to previously described groES and groEL genes and proteins. The size of the groESL-specific transcript (2.3 kb) suggested that groES and groEL of C. crescentus are organized in a bicistronic operon. Heat-shock induction of groESL mRNA is not transient, high levels of the transcript can be observed after 2 h at 40 degrees C. Primer extension experiments showed that transcription initiated at two sites. Only the start site closer to the groES coding region was highly induced during heat shock. The promoter corresponding to the heat-shock-inducible transcript has -10 and -35 regions very similar to Escherichia coli sigma 32 promoters. At normal temperatures, transcription of the groESL operon is cell-cycle controlled and both transcripts increase co-ordinately in pre-divisional cells. Transcription fusions with a lacZ reporter gene and deletions within the promoter region of the groESL operon have shown that no sequences upstream of the heat-shock promoter are necessary for temporal control. An 11 bp inverted repeat, located between the heat-shock promoter and the translation start site of groES and very similar to inverted repeats found in front of several heat-shock genes of other bacteria, may play a role in cell-cycle control of C. crescentus groESL expression.