INTRODUCTION

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The  subunit confers promoter recognition ability upon bacterial RNA polymerases. Sigma factors specifically interact with the catalytic, four-subunit core RNA polymerase (₂'). Only after assembly with  to form the so-called holo-RNA polymerase can the enzyme recognize promoter sequences and initiate transcription accurately. Upon initiation, the  factor is released and the core enzyme elongates the RNA chain (reviewed in reference 9).

Bacteria often contain multiple sigma factors (for reviews, see references 8, 10, and 11). Under normal growth conditions, transcription of most genes in Escherichia coli is initiated by RNA polymerase containing the major sigma factor ⁷⁰ (also called ^D). Alternative sigma factors coordinate transcription of distinct subsets of coregulated genes which, for example, are required for nitrogen assimilation (⁵⁴ [^N]), for flagellum gene expression (²⁸ [^F]), during stationary phase (³⁸ [^S]), or under conditions of heat stress (either ³² [^H] or ²⁴ [^E], depending on whether the stress signal is elicited in the cytoplasm or the periplasm) (17).

We are studying the regulation of heat shock genes in Bradyrhizobium japonicum, the nitrogen-fixing root nodule symbiont of the soybean plant. This organism uses three different strategies to control the expression of heat shock proteins (Hsps) at elevated temperatures. Two mechanisms rely on conserved DNA elements (CIRCE and ROSE) which are located immediately downstream of the transcription start sites of some heat shock operons (2, 18, 20). In both cases, it is likely that a putative repressor protein binds to these sites and prevents transcription under normal growth conditions. Regulation of a third class of heat shock genes is mediated by ³²-like factors (2, 16). B. japonicum is the first known organism which contains an rpoH gene family coding for three different ³²-like factors (RpoH₁, RpoH₂, and RpoH₃) (18, 19). Notably, these proteins differ not only in their primary sequence (the number of identical amino acids varies between 49 and 66%) but also in their significance to survival and in the way they are regulated. RpoH₂ appeared to be crucial for survival under all conditions because several attempts to disrupt the corresponding gene failed. By contrast, RpoH₁ and RpoH₃ could be deleted individually. Even the rpoH₁-rpoH₃ double mutant had no obvious growth defect under various conditions.

All three RpoH proteins were capable of producing high levels of -galactosidase from a groE-lacZ fusion in a ³²-deficient E. coli strain (19). In fact, rpoH₁ had originally been discovered by searching for this phenotype in a complementation approach (18). A puzzling observation was made when the abilities of the RpoH factors to complement the temperature-sensitive phenotype of the rpoH mutant were compared. Only RpoH₂ reached a similar complementation capacity to E. coli ³² in allowing the strain to grow at otherwise restrictive temperatures (19). While RpoH₁ still permitted growth at intermediate temperatures, RpoH₃ almost completely failed. Taken together, these results could be taken as a hint of a somewhat overlapping but also divergent promoter specificity of the three RpoH factors of B. japonicum.

In the present study, we set out to systematically test the promoter specificities of the RpoH factors. To complete our previous studies, we checked which Hsps were produced in each complemented E. coli strain. We were particularly interested in establishing an in vitro transcription system to approach this question. By the successful use of purified components, we show that RpoH₁ and RpoH₂ indeed favor different promoters.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. E. coli cells were grown in Luria-Bertani (LB) medium (15) supplemented with ampicillin (200 µg/ml) or kanamycin (30 µg/ml) if required. E. coli A7448 (4, 30) was grown at 28°C; the routine growth temperature for other E. coli strains was 37°C. B. japonicum was grown aerobically at 28°C in PSY medium (22) supplemented with 0.1% (wt/vol) arabinose and 100 µg of spectinomycin per ml.

Plasmid constructions. Recombinant DNA techniques were performed by standard methods (23). The individual rpoH genes were amplified from plasmids containing these genes (18, 19) by PCR with Vent DNA polymerase as specified by the manufacturer (New England Biolabs, Beverly, Mass.). The oligonucleotides used as primers were designed so that they introduced a NdeI recognition site overlapping the start codon of each gene and either a XhoI site immediately downstream of the 3' ends of rpoH₁ and rpoH₂ or a NotI site at the equivalent position of rpoH₃. The PCR-generated fragments were cut with NdeI-XhoI or NdeI-NotI and ligated into pET24b(+) (Novagen, ams Biotechnology, Lugano, Switzerland) digested with the same enzymes. The resulting plasmids, pRJ5086, pRJ5101, and pRJ5102, encoded C-terminally histidine-tagged RpoH₁, RpoH₂, and RpoH₃ proteins, respectively. The vector-encoded amino acids leucine and glutamic acid were introduced between the carboxy-terminal amino acid of RpoH₁ or RpoH₂ and the histidine tag. A stretch of five amino acids (AAALE) was introduced at the corresponding position of RpoH₃-His₆.

Overproduction and purification of RpoH-His₆ proteins. Freshly transformed E. coli BL21/pLys cells (26) carrying pRJ5086, pRJ5101, or pRJ5102 were used for overproduction of RpoH proteins. When the cells had reached an optical density at 600 nm of 0.7 at 28°C, production of the recombinant proteins was induced by the addition of 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG). After 2 h, the cells were harvested and disrupted in a French pressure cell, and proteins were purified from the soluble fraction of a 500-ml cell culture (RpoH₁ and RpoH₂) or 2,000-ml culture (RpoH₃). The extracts were loaded onto a 1.5-ml Ni-nitrilotriacetic acid-agarose column (Qiagen, Hilden, Germany), and chromatography was performed as specified in the pET System Manual (Novagen). Protein fractions were dialyzed against storage buffer (20 mM Tris-HCl [pH 8.0], 200 mM NaCl, 50% glycerol) and then stored at 20°C.

Purification of B. japonicum RNA polymerase core enzyme. RNA polymerase was prepared as described previously (3). Core RNA polymerase was separated from holoenzyme on a Resource Q column (6 ml; Pharmacia, Uppsala, Sweden). Up to 1.4 mg of RNA polymerase was loaded onto the column equilibrated with T₅₀GED (50 mM Tris-HCl [pH 8.0], 10% glycerol, 1 mM EDTA, 0.1 mM dithiothreitol) containing 0.1 M NaCl. After the column was washed with the same buffer, bound protein was eluted with 170 ml of a linear 0.1 to 0.325 M NaCl gradient. Peak fractions which were devoid of sigma factor (judged by sodium dodecyl sulfate [SDS]-polyacrylamide gel electrophoresis [12% polyacrylamide]) were pooled, dialyzed against T₁₀GED buffer containing 0.02 M NaCl and 50% (vol/vol) glycerol, and concentrated by ultrafiltration.

Western blot (immunoblot) analysis. Crude extracts of E. coli cells were prepared, separated on SDS-12% polyacrylamide gels, and transferred to nitrocellulose membranes as described previously (18). The following antisera were used: anti-E. coli ³², DnaK, and GrpE sera (B. Bukau, Freiburg, Germany; 3,000-fold dilutions [7]); anti-E. coli GroEL serum (Epicentre Technologies, Madison, Wis.; 20,000-fold dilution); and anti-E. coli IbpA serum, which recognizes the small Hsps IbpA and IbpB (A. Easton, St. Louis, Mo.; 1,500-fold dilution [1]). The primary rabbit antibodies were detected with a chemiluminescence Western blotting kit (Boehringer GmbH, Mannheim, Germany).

In vitro transcription. Multiple-round transcription assays with RNA polymerase holoenzyme were carried out in a volume of 20 µl under standard conditions as described previously (3). RNA polymerase core enzyme (1.15 µg of B. japonicum enzyme or 1 U of E. coli enzyme [Boehringer Mannheim] per assay) was reconstituted with RpoH factors (0.1 µg per assay; molar ratio of core RNA polymerase and RpoH protein, 1:1) for 20 min at 4°C in a volume of 10 µl containing 40 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 0.1 mM EDTA, 0.1 mM dithiothreitol, 150 mM KCl, 0.4 mM K₃PO₄, and 0.2 mg of bovine serum albumin per ml. The reaction was started by adding the reconstituted enzyme to a 10-µl sample containing the same components as the reconstitution buffer plus 1 mM of each nucleoside triphosphate, 1 µCi of [-³²P]UTP (800 Ci/mmol; DuPont, Bad Homburg, Germany), 1 U of RNase inhibitor per µl, and 20 nM DNA template (final concentrations).

Transcript mapping. RNA isolation and primer extension analysis was performed as described elsewhere (2). The start site of transcripts synthesized in vitro was determined as described previously (3). The oligonucleotides 702 (2) and DnaK12 (16) were used to determine the start sites of in vitro-synthesized B. japonicum groESL₁ or dnaK transcripts. Oligonucleotide Sig123, which was used to amplify the promoter region, was also used to determine the in vivo and in vitro start site of the E. coli groESL operon.

	RESULTS

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Plasmid-encoded RpoH proteins of B. japonicum selectively transcribe heat shock promoters in E. coli. We have previously described that all three RpoH proteins of B. japonicum were functionally active in a ³²-negative E. coli reporter strain which contains a chromosomally integrated E. coli groE-lacZ fusion (19). This strain produced high -galactosidase activity when it was complemented with any of the three plasmids harboring an rpoH gene under the control of the lac promoter. We now monitored the ability of these complemented strains to produce heat shock proteins in E. coli. To this end, we performed immunoblot analyses with antibodies raised against different Hsps. Extracts from normally grown and heat-shocked cells of E. coli MC4100 and the ³²-negative strain containing pUC18 without insert served as controls. The typical heat-inducible synthesis of the Hsps GroEL, DnaK, GrpE, and IbpA plus IbpB was observed in the wild type but not in the mutant (Fig. 1). As expected from the activation of the groE-lacZ fusion, all strains complemented by rpoH produced high levels of GroEL. However, the amounts of other Hsps in these strains were quite different. While E. coli ³² and B. japonicum RpoH₂ produced comparable, high levels of DnaK, GrpE, and small Hsps, RpoH₁ and in particular RpoH₃ produced only small or undetectable amounts of DnaK and GrpE. This result indicated that the sigma factors conferred different promoter specificities upon E. coli core RNA polymerase.

View larger version (80K): [in this window] [in a new window]   FIG. 1.   Detection of heat shock proteins produced by E. coli MC4100 (WT) and E. coli A7448 (rpoH). E. coli A7448 transformed with pUC18 (), pRJ5000 (rpoH1, indicated as 1), pRJ5002 (rpoH2, indicated as 2), pRJ5040 (rpoH3, indicated as 3), or pEC5007 (E. coli rpoH, indicated as E) was grown in the presence of 0.5 mM IPTG. Crude cell extracts were prepared from cells grown at 28°C (0) or heat-shocked cells (heat shock [HS] from 28 to 43°C for 5 or 10 min as indicated) and separated on a SDS-12% polyacrylamide gel. A Coomassie blue-stained gel is shown at the top. The apparent molecular masses (in kilodaltons) of relevant marker proteins are indicated to the left of the gel. Similar gels with suitably diluted samples (1/5, 1/10, 1/3, and 1/15 of the amounts loaded onto the stained gel) were used for immunodetection of the Hsps DnaK, GroEL, GrpE, and IbpA plus IbpB, respectively.

The B. japonicum RpoH factors use the same in vivo start site as E. coli ³². Since all four RpoH factors synthesized GroEL protein efficiently, we analyzed whether the heterologous proteins initiated transcription at the same position as E. coli ³². Total RNA was isolated from each complemented strain, and the 5' end of the groESL transcript was determined by primer extension. This strain contains two groESL promoter regions: the original operon and a chromosomally integrated groE-lacZ fusion. Transcripts of both operons were detected by the primer used in these experiments. The transcripts formed by all four RpoH proteins start at the identical position, which corresponds to the previously described ³²-dependent site (Fig. 2) (5). Small amounts of a shorter product probably represent prematurely terminated reverse transcripts. RNA from the mutant containing only the vector resulted in a faint signal that represents transcripts originating from the ⁷⁰-dependent promoter (Fig. 2) (30). This transcript was hardly observed with RNA from the rpoH⁺ strains, presumably because the abundant RpoH proteins prevented transcription from the ⁷⁰ promoter by promoter occlusion. It has been shown that this promoter is very weak and virtually inactive in rpoH⁺ strains (14, 30).

View larger version (80K): [in this window] [in a new window]   FIG. 2.   Primer extension analysis of groESL transcripts synthesized in E. coli A7448. Reverse transcripts were obtained with 15 µg of total RNA isolated from the strain complemented with B. japonicum rpoH1 (lane 1), rpoH2 (lane 2), or rpoH3 (lane 3) or E. coli rpoH (lane E) and 30 µg of RNA from the same strain transformed with pUC18 (lane ). Oligonucleotide Sig123 was used as the primer for the primer extension and corresponding sequencing reactions (lanes T, C, G, and A). The transcription start site used by the 32-like proteins is indicated by a solid arrow; the open arrow marks the transcript initiated at the 70-dependent promoter.

Overexpression and purification of histidine-tagged RpoH₁, RpoH₂, and RpoH₃. Knowing that amino-terminally or carboxy-terminally histidine-tagged ³² of E. coli is active in vivo and in vitro (7), we constructed plasmids to produce the B. japonicum RpoH proteins with a histidine tag attached to their C termini. Crude cell extracts containing these proteins were prepared and separated into pellet and supernatant fractions (Fig. 3). Surprisingly, the overproduction efficiency and the solubility of the RpoH proteins varied over a wide range. RpoH_2His and RpoH_3His were overproduced to the highest levels. However, more than 95% of RpoH_3His was found in the pellet fraction, indicating that the majority of this protein was insoluble. By contrast, approximately 50% of overproduced RpoH_1His and RpoH_2His remained in the supernatant. All three proteins were purified from the soluble supernatant fraction by nickel-nitrilotriacetic acid chromatography. The proteins exhibited different binding affinities to the column resin. While some RpoH_2His had already eluted during the wash step with an imidazole concentration of 50 mM, RpoH_1His and RpoH_3His were still retained at this concentration (data not shown). The purification profile for RpoH_2His (Fig. 4A) serves as an example to show that the nickel affinity chromatography technique allowed efficient purification of histidine-tagged B. japonicum RpoH proteins.

View larger version (85K): [in this window] [in a new window]   FIG. 3.   Solubility of overproduced B. japonicum RpoH proteins. The supernatant (Sup) and pellet (Pellet) fraction of crude cell extracts prepared before () or after (+) IPTG induction were separated by centrifugation at 15,000 rpm (SS-34 rotor) at 4°C for 30 min. Control cells contain the vector pET24b(+) without insert. RpoH1His, RpoH2His, and RpoH3His were produced from plasmids pRJ5086, pRJ5101, and pRJ5102, respectively. After electrophoresis in an SDS-12% polyacrylamide gel, the proteins were stained with Coomassie blue. The apparent molecular masses (in kilodaltons) of marker proteins are indicated to the left of the gel. Arrows point to the overproduced RpoHHis protein.

View larger version (61K): [in this window] [in a new window]   FIG. 4.   Purification of RpoH2His and immunodetection of B. japonicum RpoHHis proteins. (A) Aliquots of the RpoH2His supernatant fraction which was loaded onto the column (load), the flowthrough (flow), the different wash (wash), and the elution (eluate) fractions were separated on an SDS-12% polyacrylamide gel, and the proteins were stained with Coomassie blue. The imidazole concentration in the wash and elution buffers is indicated. The apparent molecular masses (in kilodaltons) of the marker proteins are indicated to the left of the gel. The arrow points to the protein fraction which was subsequently used for in vitro experiments. (B) Coomassie blue-stained SDS-12% polyacrylamide gel of 100 ng of each purified B. japonicum RpoHHis protein. The apparent molecular masses of two marker proteins are indicated to the right of the gel. (C) Immunodetection of purified B. japonicum RpoH proteins with anti-E. coli 32 serum. For each protein, a 5-ng sample was subjected to Western blot analysis.

Multiple-round in vitro transcription assays. In a first attempt to test whether the purified RpoH proteins were transcriptionally active in vitro, we simulated the situation in the E. coli rpoH mutant in which the proteins had been shown to function in vivo. E. coli core RNA polymerase was reconstituted with the individual RpoH_His proteins, and the ability to transcribe the E. coli groESL promoter was monitored in a multiple-round transcription assay. The core enzyme alone produced small amounts of transcript (Fig. 5A), most probably as a result of some residual ³² protein in the preparation that was detectable by Western blot analysis (data not shown). Transcription was stimulated by the addition of RpoH_1His and in particular by RpoH_2His. RpoH_3His appeared to be inactive because the reconstituted enzyme did not produce more transcript than core RNA polymerase alone. Since RpoH₃ was active at the E. coli groESL promoter in vivo (compare Fig. 1) we conclude that the purified protein was in an inactive state, probably because the small portion of soluble protein was in an inactive conformation. Transcriptional activity of RpoH₁ and RpoH₂ required the presence of RNA polymerase because the sigma factors alone were unable to synthesize transcript. As positive controls, we used E. coli and B. japonicum holo-RNA polymerases, both of which contain significant amounts of RpoH protein as revealed by Western blot analysis (data not shown). The transcripts produced by these enzymes were indistinguishable from the ones formed by the reconstituted enzymes, indicating that transcription had been initiated at the same position (Fig. 5A).

View larger version (60K): [in this window] [in a new window]   FIG. 5.   Transcription of the E. coli groESL promoter by E. coli RNA polymerase core enzyme and B. japonicum RpoH factors. (A) The enzyme combinations tested are indicated. Core, E. coli RNA polymerase core enzyme; RpoH, purified RpoHHis protein; Ec holo, E. coli RNA polymerase holoenzyme; Bj holo, B. japonicum RNA polymerase holoenzyme. (B) Primer extension analysis of in vitro-synthesized RNA. The enzyme with which the transcript was generated is indicated. The primer extension and sequencing reactions (TCGA) were performed with oligonucleotide Sig123. The transcription start site is depicted by an arrow.

View larger version (51K): [in this window] [in a new window]   FIG. 6.   Transcription of the B. japonicum groESL1 promoter and the dnaKJ promoter by B. japonicum RNA polymerase reconstituted with purified RpoH factors. (A) Transcription of the B. japonicum groESL1 promoter. The enzyme combinations tested are indicated. Core, B. japonicum RNA polymerase core enzyme; RpoH, purified RpoHHis protein. The arrow points to an RNA size marker of 406 nucleotides. (B) Transcription of the B. japonicum dnaKJ promoter. The enzyme combinations are as indicated in panel A. (C) Primer extension analysis of in vitro-synthesized groESL1 transcript. Oligonucleotide 702 was used for the sequencing (TCGA) and primer extension reaction. The triangle points to the start site, which has been determined in vivo (2). The origin of small amounts of slower-migrating bands is not known because corresponding products were not observed after in vitro transcription. (D) Primer extension analysis of in vitro-synthesized B. japonicum dnaKJ transcript. Oligonucleotide DnaK12 was used for the sequencing (TCGA) and primer extension reaction. The 5' end of the reverse transcript (indicated by a triangle) corresponds to the major start site found in vivo (16).

Single-round in vitro transcription assays. To gain more insight into possible promoter specificities of RpoH₁ and RpoH₂, it was important to establish a single-round transcription system. B. japonicum core RNA polymerase was reconstituted with RpoH_2His, and transcription at five different promoters was assayed under conditions which allowed for the production of only one transcript by each preformed RNA polymerase-template complex. First we tested the E. coli groESL promoter, which was transcribed very well by this enzyme combination (Fig. 7A). The B. japonicum dnaKJ and groESL₁ promoters were also transcribed specifically (Fig. 7B). As a control, we examined the hspA rpoH₁ and rrn promoters, which are expected to be transcribed by B. japonicum ⁸⁰, the ⁷⁰ homolog (3, 18). The corresponding products would be 625 and 240 nucleotides long, respectively. Neither promoter was transcribed by the RNA polymerase containing RpoH_2His, indicating that this sigma factor specifically recognizes ³²-like promoters.

View larger version (51K): [in this window] [in a new window]   FIG. 7.   Single-round transcription of different promoters by B. japonicum core RNA polymerase reconstituted with RpoH2His. The E. coli (A) or B. japonicum (B) promoters which were transcribed are indicated. Experiments were performed with core RNA polymerase alone () and the same enzyme reconstituted with RpoH2His (+). Arrows point to the transcripts; the position and length (in nucleotides) of RNA size markers are indicated to the right.

Promoter competition experiments. To further investigate the promoter specificities of RpoH₁ and RpoH₂, we performed experiments in which an equimolar mix of different templates was provided for transcription. The first mix contained a composite of all five promoters which had been tested individually in the last experiment. B. japonicum core RNA polymerase was reconstituted with RpoH_1His and RpoH_2His, and transcription was assayed under single-round conditions (Fig. 8). As suggested by the experiment in Fig. 7, RNA polymerase containing RpoH_2His preferentially transcribed from the E. coli groESL promoter. (The preference for the E. coli promoter may even be underestimated, because the E. coli groESL transcript contains 50 uracils that can be radiolabeled whereas the B. japonicum dnaKJ and groESL₁ transcripts contain 81 and 80 uracils, respectively.) Transcription from the B. japonicum ³²-type promoters was much lower with the dnaKJ promoter, yielding slightly more transcript than the groESL₁ promoter. RNA polymerase containing RpoH_1His also transcribed the E. coli groESL promoter to the highest level. Small amounts of the groESL₁ transcript and very little dnaKJ transcript were obtained, which implies that RpoH₁ and RpoH₂ have different promoter specificities. The hspA rpoH₁ and rrn promoters that were also present in the mixture were not transcribed by the reconstituted enzymes.

View larger version (63K): [in this window] [in a new window]   FIG. 8.   Single-round promoter competition experiment with B. japonicum core RNA polymerase reconstituted with RpoH1His or RpoH2His. The E. coli groESL promoter and the B. japonicum dnaKJ, groESL1, hspA rpoH1, and rrn promoters were provided at equimolar concentrations (10 nM each; the template concentration is not limiting in this assay). The transcripts obtained are indicated.

View larger version (68K): [in this window] [in a new window]   FIG. 9.   Single-round promoter competition experiment with B. japonicum RNA polymerase core enzyme and holoenzyme reconstituted with purified RpoH proteins. The B. japonicum dnaKJ, groESL1, hspA rpoH1, and rrn promoters were provided at equimolar concentrations (10 nM each). The corresponding transcripts are indicated. The vector-encoded reference transcript (ref) was produced only in the presence of the holoenzyme.

	DISCUSSION

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Different promoter specificities of B. japonicum RpoH factors. After the previous identification of three disparately regulated rpoH genes in B. japonicum (18, 19), we were left with the question of why this organism uses a gene family for functions that in other bacteria (e.g., E. coli) are accomplished by a single rpoH gene. In principle, an organism can use two different strategies to achieve coordinate regulation of a gene under different environmental conditions: (i) The gene can be put under the control of different promoters, or (ii) several copies of this gene may be present, each having a distinctly regulated promoter. A paradigm for the differential regulation of reiterated genes is the groESL multigene family of B. japonicum. The expression of five groESL operons appears to be controlled by four mechanisms, two of which do not contribute to chaperonin production under heat shock conditions (low constitutive expression of groESL₂ and NifA-dependent expression of groESL₃) (6). Heat-induced expression of groESL genes depends on two separate mechanisms, namely, RpoH factor(s) for groESL₁ and CIRCE (controlling inverted repeat of chaperone expression) (31) for groESL₄ and groESL₅ (2). The rpoH genes of B. japonicum were also defined as a disparately regulated gene family. Transcription of rpoH₁, for instance, is heat inducible by a novel mechanism (18, 20), whereas rpoH₂ is transcribed constitutively from a ⁷⁰-type promoter under normal growth conditions and induced from a ^E-type promoter under severe heat shock conditions (19).

Function of RpoH₁ and RpoH₂ in B. japonicum. On the basis of results obtained previously it was proposed that RpoH₂ is essential for the synthesis of ³²-dependent proteins under normal growth conditions whereas RpoH₁ provides their synthesis under stress conditions (19). The function of RpoH₃ remains obscure. Combining all data known at present, we arrive at the following, refined model.

(i) Normal growth conditions. Although the rpoH₂ gene is transcribed to a considerable extent under these conditions, very little RpoH protein is detectable in cell extracts (18, 19). The corresponding protein is probably unstable during normal growth, as is E. coli ³² (25). The observation that neither the rpoH₂ nor the dnaK gene could be deleted had already suggested a direct dependence of the DnaK chaperone, which is required under all conditions, on this sigma factor (16, 19). The in vitro transcription data confirm that RpoH₂ directs the transcription of the dnaKJ promoter efficiently. The groESL₁ promoter could also be transcribed in vitro, but this does not seem to play a role in vivo because the groESL₁ transcript is almost undetectable under normal growth conditions (2).

(ii) Heat shock conditions. A temperature upshift greatly stimulates the production of RpoH₁ in B. japonicum (18, 19). The parallel induction of the groESL₁ transcript (2) indicated that it occurred as a result of an increase in RpoH₁ production. The in vitro activity of RpoH₁ supports this notion. By comparison, the dnaKJ promoter was weakly transcribed, which raises the question how heat induction of DnaK is accomplished. Since maximal induction of this protein is already reached a few minutes after a temperature upshift (16), it is highly possible that RpoH₂ is responsible for this increase. The RpoH₂ level in the cell increases in the initial phase after a heat shock before it rapidly decreases (19). Under continuous stress conditions, the weak activity of RpoH₁ may then suffice to keep the DnaK concentration at an elevated level.