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RpoH ( Escherichia coli ς 32 and its homologs) is the central regulator of the heat shock response in gram-negative proteobacteria. Here we studied salient regulatory features of RpoH in Agrobacterium tumefaciens by examining its synthesis, stability, and activity while increasing the temperature from 25 to 37°C. Heat induction of RpoH synthesis occurred at the level of transcription from an RpoH-dependent promoter, coordinately with that of DnaK, and followed by an increase in the RpoH level. Essentially normal induction of heat shock proteins was observed even with a strain that was unable to increase the RpoH level upon heat shock. Moreover, heat-induced accumulation of dnaK mRNA occurred without protein synthesis, showing that preexisting RpoH was sufficient for induction of the heat shock response. These results suggested that controlling the activity, rather than the amount, of RpoH plays a major role in regulation of the heat shock response. In addition, increasing or decreasing the DnaK-DnaJ chaperones specifically reduced or enhanced the RpoH activity, respectively. On the other hand, the RpoH protein was normally stable and remained stable during the induction phase but was destabilized transiently during the adaptation phase. We propose that the DnaK-mediated control of RpoH activity plays a primary role in the induction of heat shock response in A. tumefaciens , in contrast to what has been found in E. coli .

In the paradigm of bacterial heat shock response studied with Escherichia coli, ς32 (encoded by rpoH) plays a key role in controlling the transcription of all major heat shock proteins (HSPs) in the cytoplasm, including GroE and DnaK chaperone machineries and ATP-dependent proteases, such as Clp and Lon (10, 42). Upon heat shock, the cellular level of ς32 increases rapidly both by enhanced translation of rpoH mRNA and by transient stabilization of ς32, and this increase in turn activates the transcription of heat shock genes. Homologs of rpoH have been identified from some 20 species of eubacteria that belong to the alpha, beta, and gamma subgroups of proteobacteria (1, 13, 15, 20, 21, 24, 30, 31); earlier work cited in reference (21). The rpoH homologs from many gamma proteobacteria share common structural features with E. coli rpoH, a downstream box, mRNA secondary structure, and highly conserved amino acid sequence of region C, that are important for thermoregulation of rpoH translation and ς32 stability and activity in E. coli (21). In these bacteria, the RpoH homologs indeed exhibit translational induction and stabilization upon heat shock very similar to that found with E. coli (22). In contrast, alpha and beta proteobacteria have diverged from the gamma subgroup in their modes of regulation of rpoH expression. Their rpoH genes do not contain the downstream box or mRNA secondary structure that are conserved among gamma proteobacteria. Instead, some rpoH genes from alpha proteobacteria contain an RpoH-dependent promoter that can be induced upon heat shock (24, 26, 40). Another important feature found in alpha but not in gamma proteobacteria is the presence of the CIRCE (for controlling inverted repeat of chaperone expression)-HrcA regulatory system. Recent studies of wide groups of eubacteria revealed a variety of heat shock regulatory mechanisms, either positive regulation by alternative ς factors or negative regulation by specific repressor-operator systems (see reviews in references 23 and 28). The most widely distributed system is the CIRCE-HrcA system, extensively characterized in Bacillus subtilis as the regulatory mechanism specific for the groE and dnaK operons (18, 19, 41, 43). CIRCE, with a consensus of TTAGCACTC-N9-GAGTGCTAA, represents a site for binding the HrcA repressor. HrcA could act as a stress sensor whose activity is modulated by the GroEL chaperone. This system has been found in gram-positive bacteria and proteobacteria and implicated in cyanobacteria and several other groups of eubacteria (5, 8, 9, 38, 39). Within proteobacteria, CIRCE and/or hrcA were shown to regulate groE operons of some alpha proteobacteria, including Agrobacterium tumefaciens (3, 27, 37). However a CIRCE-like sequence has not been detected in gamma proteobacteria, except for the groE operon of Chromatium vinosum (7). It seemed likely that the CIRCE-HrcA system is operative in the alpha and beta proteobacteria but not in most gamma proteobacteria. Thus, alpha and beta proteobacteria appeared to be unique in that they carry both the RpoH system, with regulatory features different from those of gamma proteobacteria, and the CIRCE-HrcA system for regulation of the heat shock response. In this study, we used A. tumefaciens as a model to study the differential roles of the RpoH and CIRCE-HrcA systems in the heat shock response in alpha proteobacteria. The groE and dnaK operons of A. tumefaciens were previously isolated and characterized (34–37), and the CIRCE element was found in the groE, but not in the dnaK, promoter region. Although the rpoH gene was available from our previous study (21), no information on hrcA was at hand. Successful isolation of hrcA and analysis of the deletion mutants lacking rpoH and/or hrcA revealed that RpoH plays a major and global role in the induction of HSPs, whereas the role of HrcA is restricted primarily to repressing the groE operon under nonstress conditions.

The heat shock response in Escherichia coli depends primarily on the increased synthesis and stabilization of otherwise scarce and unstable ς 32 ( rpoH gene product), which is required for the transcription of heat shock genes. The heat-induced synthesis of ς 32 occurs at the level of translation, and genetic evidence has suggested the involvement of a secondary structure at the 5′ portion (nucleotides −19 to +247) of rpoH mRNA in regulation. We now present evidence for the mRNA secondary structure model by means of structure probing of RNA with chemical and enzymatic probes. A similar analysis of several mutant RNAs with a mutation predicted to alter a base pairing or with two compensatory mutations revealed altered secondary structures consistent with the expression and heat inducibility of the corresponding fusion constructs observed in vivo. These findings led us to assess the possible roles of each of the stem-loop structures by analyzing an additional set of deletions and base substitutions. The results indicated not only the primary importance of base pairings between the translation initiation region of ca. 20 nucleotides (the AUG initiation codon plus the “downstream box”) and the internal region of rpoH mRNA but also the requirement of appropriate stability of mRNA secondary structures for characteristic thermoregulation, i.e., repression at a low temperature and induction upon a temperature upshift.

Induction of heat shock proteins represents a ubiquitous and homeostatic cellular response to cope with the accumulation of unfolded and misfolded proteins following exposure to heat or other stresses. In Escherichia coli, the induction occurs primarily by activating the transcription of heat shock genes by a transient increase in the cellular level of ς32 (10), encoded by the rpoH gene (8, 36). The level of ς32 during steady-state growth is very low (ca. 10 to 30 molecules per cell at 30°C [4]) due to the extreme instability (half life, 1 min) and restricted translation of rpoH mRNA (8, 37). Upon exposure to higher temperatures (e.g., 42°C), both rapid stabilization of ς32 and translational activation of rpoH mRNA result in a marked and transient increase in the ς32 level, which in turn activates the transcription of heat shock genes such as those encoding molecular chaperones (e.g., DnaK and GroEL) and ATP-dependent proteases (e.g., Lon and ClpP) (7, 8). Whereas stabilization of ς32 is thought to occur by accumulation of partially unfolded proteins that can titrate chaperones away from ς32 (2, 4), translational activation probably occurs by resolving extensive mRNA secondary structure formed under nonstress conditions (17, 37, 38). Previous work on heat-induced synthesis of ς32–β-galactosidase fusion protein in E. coli revealed that two proximal coding regions (A and B) of rpoH mRNA are involved in translational control of ς32 synthesis (17, 37). Region A is similar to the “downstream box,” which is complementary to part of 16S rRNA (27), and acts as a positive element by enhancing translation, whereas region B is a negative element which forms an extensive secondary structure with region A. In addition, a segment of ς32 (region C; around residues 122 to 144 [18]) that corresponds to an area further downstream of rpoH was shown to be important for the DnaK/DnaJ-mediated negative feedback control of the heat shock response (28, 30, 31). Region C affects both the synthesis and stability of the fusion protein (18, 37), presumably by providing sites for interaction with DnaK (14). Recent work in this and other laboratories led to the isolation and characterization of rpoH genes encoding ς32 (RpoH) homologs from a number of gram-negative bacteria (1, 5, 6, 16, 19–23, 25, 34). Some of them were isolated by directly selecting for clones that can complement the defects of an E. coli ΔrpoH mutant which grows only at or below 20°C (1, 19, 21). Comparison of nucleotide and amino acid sequences among these homologs revealed several conserved sequences that seemed to be of regulatory importance for the heat shock response. Specifically, both downstream box and predicted mRNA secondary structures similar to those seen in E. coli were found among the homologs from most members of the gamma but not alpha proteobacteria (1, 19, 34, 36). Besides, a stretch of 9 amino acid residues, highly conserved among all ς32 homologs but absent in other ς factors (designated the RpoH box [19, 36]), was detected within region C, implicated in the chaperone-mediated feedback control (18; see above). These interesting developments prompted us to examine the regulatory roles of ς32 homologs in the heat shock response of some of the representative bacteria. In this report, we analyzed the regulatory and functional properties of ς32 homologs (RpoH) both in E. coli and in cognate bacteria. Several members of the gamma proteobacteria (E. coli, Serratia marcescens, Proteus mirabilis, and Pseudomonas aeruginosa) and one of the alpha proteobacteria (Agrobacterium tumefaciens) were chosen for this study. The ability of these rpoH homologs to complement, at least partially, the growth defect of the E. coli ΔrpoH mutant had suggested that they can be expressed to appreciable extents in E. coli and can functionally replace ς32 to varying degrees. Such expectations were fully supported by the present results. Most significantly, they revealed substantial conservation and some divergence in the regulatory strategy of ς32 homologs, particularly among the gamma proteobacteria.

Escherichia coli mutants lacking activities of all known cytosolic ATP-dependent proteases (Lon, ClpAP, ClpXP, and HslVU), due to double deletions [Δ hslVU and Δ( clpPX-lon )], cannot grow at low (30°C) or very high (45°C) temperatures, unlike those carrying either of the deletions. Such growth defects were particularly marked when the deletions were introduced into strain MG1655 or W3110. To examine the functions of HslVU and other proteases further, revertants that can grow at 30°C were isolated from the multiple-protease mutant and characterized. The revertants were found to carry a suppressor affecting either ftsZ (encoding a key cell division protein) or sulA (encoding the SulA inhibitor, which binds and inhibits FtsZ). Whereas the ftsZ mutations were identical to a mutation known to produce a protein refractory to SulA inhibition, the sulA mutations affected the promoter-operator region, reducing synthesis of SulA. These results suggested that the growth defect of the parental double-deletion mutant at a low temperature was due to the accumulation of excess SulA without DNA-damaging treatment. Consistent with these results, SulA in the double-deletion mutant was much more stable than that in the Δ( clpPX-lon ) mutant, suggesting that SulA can be degraded by HslVU. As expected, purified HslVU protease degraded SulA (fused to the maltose-binding protein) efficiently in an ATP-dependent manner. These results suggest that HslVU as well as Lon participates in the in vivo turnover of SulA and that HslVU becomes essential for growth when the Lon (and Clp) protease level is reduced below a critical threshold.