Downregulation of the heat shock response is independent of DnaK and σ<sup>32</sup> levels in <i>Caulobacter crescentus</i>

Silva, Antonio C. A. Da; Simao, T. Rita C. G.; Susin, Michelle F.; Baldini, Regina L.; Avedissian, Marcelo; Gomes, Suely L.

doi:10.1046/j.1365-2958.2003.03581.x

Cited by 32 publications

(44 citation statements)

References 47 publications

(81 reference statements)

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“…The rpoH genes from Caulobacter crescentus (51), Rhodobacter capsulatus (10), and Agrobacterium tumefaciens (33) are positively autoregulated from an RpoH-dependent promoter upon heat shock. In C. crescentus, down-regulation of the stress response is independent of DnaK and RpoH (9). In A. tumefaciens, DnaK-dependent regulation of RpoH has also been shown to occur at the level of activity (34).…”

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The RpoH-Mediated Stress Response in Neisseria gonorrhoeae Is Regulated at the Level of Activity

et al. 2004

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The general stress response in Neisseria gonorrhoeae was investigated. Transcriptional analyses of the genes encoding the molecular chaperones DnaK, DnaJ, and GrpE suggested that they are transcribed from 32 (RpoH)-dependent promoters upon exposure to stress. This was confirmed by mutational analysis of the 32 promoter of dnaK. The gene encoding the gonococcal RpoH sigma factor appears to be essential, as we could not isolate viable mutants. Deletion of an unusually long rpoH leader sequence resulted in elevated levels of transcription, suggesting that this region is involved in negative regulation of RpoH expression during normal growth. Transcriptional analyses and protein studies determined that regulation of the RpoH-mediated stress response is different from that observed in most other species, in which regulation occurs predominantly at the transcriptional and translational levels. We suggest that an increase in the activity of preformed RpoH is primarily responsible for induction of the stress response in N. gonorrhoeae.

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The RpoH-Mediated Stress Response in Neisseria gonorrhoeae Is Regulated at the Level of Activity

et al. 2004

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“…The overexpressing strain NA1000 pRB51 carries a multicopy plasmid in which the groESL genes are under the control of the P xylX promoter (7). Thus, in the presence of xylose the levels of GroES/GroEL are two to threefold higher than in those in the control strain NA1000 pAS22, which carries only the chromosomal copy of the groESL genes (7). As the results shown in Table 3 indicate, ␤-galactosidase activity in C. crescentus cells carrying transcription fusion pMA11 was 30% lower in cells overexpressing GroES/GroEL (NA1000 pRB51) than in cells presenting normal levels of these chaperones (NA1000 pAS22).…”

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confidence: 92%

Functional and Structural Analysis of HrcA Repressor Protein from Caulobacter crescentus

et al. 2004

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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...

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“…Given that dnaK is essential in C. crescentus (19,20), we pursued a strategy to generate a minimal functional allele of dnaK. Based on the high-density Tn sequencing data of Christen et al (20), we noted that C. crescentus tolerates Tn5 insertions at the 3= end of dnaK, after codon 496.…”

Section: Resultsmentioning

confidence: 99%

“…In contrast to E. coli, DnaK is essential for growth in C. crescentus (19). However, removing the alpha-helical lid domain at the C terminus of DnaK does not have a significant effect on C. crescentus growth; three independent genetic studies have identified viable strains bearing transposon insertions (20), frameshift mutations (34), or nonsense mutations (this work) in the linker that connect the lid domain to the substrate binding beta sheet domain.…”

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confidence: 99%

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Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK

2016

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Growth in a surface-attached bacterial community, or biofilm, confers a number of advantages. However, as a biofilm matures, high-density growth imposes stresses on individual cells, and it can become less advantageous for progeny to remain in the community. Thus, bacteria employ a variety of mechanisms to control attachment to and dispersal from surfaces in response to the state of the environment. The freshwater oligotroph Caulobacter crescentus can elaborate a polysaccharide-rich polar organelle, known as the holdfast, which enables permanent surface attachment. Holdfast development is strongly inhibited by the small protein HfiA; mechanisms that control HfiA levels in the cell are not well understood. We have discovered a connection between the essential general protein chaperone, DnaK, and control of C. crescentus holdfast development. C. crescentus mutants partially or completely lacking the C-terminal substrate binding "lid" domain of DnaK exhibit enhanced bulk surface attachment. Partial or complete truncation of the DnaK lid domain increases the probability that any single cell will develop a holdfast by 3-to 10-fold. These results are consistent with the observation that steady-state levels of an HfiA fusion protein are significantly diminished in strains that lack the entire lid domain of DnaK. While dispensable for growth, the lid domain of C. crescentus DnaK is required for proper chaperone function, as evidenced by observed dysregulation of HfiA and holdfast development in strains expressing lidless DnaK mutants. We conclude that DnaK is an important molecular determinant of HfiA stability and surface adhesion control. IMPORTANCERegulatory control of cell adhesion ensures that bacterial cells can transition between free-living and surface-attached states. We define a role for the essential protein chaperone, DnaK, in the control of Caulobacter crescentus cell adhesion. C. crescentus surface adhesion is mediated by an envelope-attached organelle known as the holdfast. Holdfast development is tightly controlled by HfiA, a small protein inhibitor that directly interacts with a WecG/TagA-family glycosyltransferase required for holdfast biosynthesis. We demonstrate that the C-terminal lid domain of DnaK is not essential for growth but is necessary for proper control of HfiA levels in the cell and for control of holdfast adhesin development. Communities of surface-attached bacteria, called biofilms, account for the majority of cells on the surface of our planet. Biofilms allow bacteria to engage in metabolic cooperation, share genetic information, and afford protection from chemical and physical stresses in the environment. Moreover, organic biopolymers and ions accumulate at surfaces through a process known as conditioning (1-4); thus, the ability of a bacterial cell to adhere can confer a nutritional advantage, particularly in oligotrophic environments where nutrients are extremely scarce (5). Clearly, the capacity to transition from a free-living, unicellular lifestyle into a surface-attached ...

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Downregulation of the heat shock response is independent of DnaK and σ³² levels in Caulobacter crescentus

Abstract: Summary Expression of heat shock genes in Gram

Cited by 32 publications

References 47 publications

The RpoH-Mediated Stress Response in Neisseria gonorrhoeae Is Regulated at the Level of Activity

The RpoH-Mediated Stress Response in Neisseria gonorrhoeae Is Regulated at the Level of Activity

Functional and Structural Analysis of HrcA Repressor Protein from Caulobacter crescentus

Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK

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Downregulation of the heat shock response is independent of DnaK and σ32 levels in Caulobacter crescentus

Abstract: Summary Expression of heat shock genes in Gram

Cited by 32 publications

References 47 publications

The RpoH-Mediated Stress Response in Neisseria gonorrhoeae Is Regulated at the Level of Activity

The RpoH-Mediated Stress Response in Neisseria gonorrhoeae Is Regulated at the Level of Activity

Functional and Structural Analysis of HrcA Repressor Protein from Caulobacter crescentus

Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK

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Downregulation of the heat shock response is independent of DnaK and σ³² levels in Caulobacter crescentus