Functional and Structural Analysis of HrcA Repressor Protein from Caulobacter crescentus

Abstract

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 32 -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.Caulobacter crescentus, a member of the ␣-subdivision of proteobacteria, responds to heat shock by elevating the levels of synthesis of over 20 polypeptides (16). Several heat shockinducible genes in C. crescentus have been characterized, including dnaKJ (15), groESL (1), lon (37), hrcA/grpE (30), and ftsH (11), and they all are positively regulated by the alternative sigma factor of RNA polymerase 32 . The rpoH gene encoding 32 in C. crescentus has also been characterized, and one of its promoters was shown to be 32 dependent, indicating an autogenous control of rpoH transcription (28, 38). Furthermore, the levels of 32 were shown to increase transiently during heat shock, and the increased transcription of rpoH seems to account for the induction of 32 levels (28, 38). Recent studies have shown that, as described for Escherichia coli (for a review, see reference 39), the heat shock protein (HSP) DnaK is a negative modulator of the heat shock response in C. crescentus, which acts by inhibiting 32 activity and stimulating its degradation (7). However, despite the strong effect of DnaK levels on the induction phase of the response, downregulation of HSP synthesis is not affected by changes in the amount of this chaperone. Competition between 32 and 73 , the major sigma factor in C. crescentus which was shown to be heat shock inducible, has been proposed as the most important factor controlling the shutoff of HSP synthesis during the recovery phase (7). Another important negative modulator of the heat shock response, the repressor protein HrcA in C. crescentus, has also been described (30). This protein, which is absent in E. coli, has been found to occur in a growing number of diverse bacterial species and pr...

Microarray-Based Characterization of theListeria monocytogenesCold Regulon in Log- and Stationary-Phase Cells

“…Interestingly, the hrcA-grpE-dnaK operon showed 3.4-to 4.3-fold-lower transcript levels in log-phase cells grown at 4°C (see Table S3 in the supplemental material). This finding was surprising as the HrcA-repressed groES-groEL operon also showed lower transcript levels, but this finding may be a consequence of posttranscriptional regulation of HrcA activity (89). cspB, which encodes a putative cold shock protein, shows 36.1-fold-lower transcript levels at 4°C than at 37°C (see Table S3 in the supplemental material), consistent with observations that the cspB homologs in E. coli and B. subtilis do not seem to be critical for growth at low temperatures (32,99).…”

A Chlamydia-Specific C-Terminal Region of the Stress Response Regulator HrcA Modulates Its Repressor Activity

“…During cellular stress, however, GroEL is titrated away to correct protein misfolding, which reduces HrcA-CIRCE binding and causes derepression of the heat shock genes. While there is evidence that GroEL promotes HrcA-CIRCE binding (23,27,37,47), the mechanism has not been determined experimentally. From in vitro studies, GroEL has been proposed to increase HrcA binding to its operator by correcting HrcA misfolding via its role as an ATP-dependent molecular chaperone (23,27).…”

“…HrcA orthologs in bacteria are generally proteins of 39 to 40 kDa in molecular size, with conservation in two N-terminal regions that make up the DNA-binding domain and a C-terminal region of unknown function (21,34,41,47). However, we noted that HrcA encoded by all Chlamydia spp.…”

“…We focused our efforts on GroEL since this heat shock protein has been shown to copurify as a complex with endogenous HrcA from a C. trachomatis lysate (53). Furthermore, GroEL has been shown to promote the binding of HrcA to CIRCE in vitro for a number of bacterial species (23,27,37,47). This interaction with GroEL has been proposed to increase the binding affinity of HrcA for CIRCE by inducing an active conformation of the repressor (27).…”

Regulação da expressão da fímbria CupD por sistemas de dois componentes de Pseudomonas aeruginosa Pa14 e ensaios de virulência no hospedeiro-modelo Dictyostelium discoideum

“…Composição Referência Tris-HCl 10mM (pH 7,9), MgCl 2 10mM, DTT 1 mM, KCl 100 mM, glicerol 10%, 1 µg DNA de esperma de salmão, com e sem 10 µg/µL de BSA Dekimpe e Déziel, comunicação pessoal Tris-HCl 10mM (pH 7,9), MgCl 2 10mM, DTT 1 mM, KCl 40 mM, glicerol 10%, 1 µg DNA de esperma de salmão, 10 µg/µL de BSA Dekimpe e Déziel, comunicação pessoal Tris-HCl 10mM (pH 7,9), MgCl 2 10mM, DTT 2 mM, NaCl 100mM, glicerol 10%, 1 µg DNA de esperma de salmão (Susin et al, 2004) Tris-HCl 10mM (pH 7,5), MgCl 2 10mM, DTT 0,1 mM, KCl 120mM, glicerol 10%, 1 µg DNA de esperma de salmão, 10 µg/µL de BSA Braz e Marques, comunicação pessoal…”