DnaK chaperone-mediated control of activity of a sigma(32) homolog (RpoH) plays a major role in the heat shock response of Agrobacterium tumefaciens

Abstract

RpoH (Escherichia coli sigma(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 degrees 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.

FIG. 1

Transient increases in the level and synthesis rate of RpoH upon heat shock. (A) Cells of wild-type A. tumefaciens (GV3101) were grown in complete medium (●) or synthetic medium (▪) to the logarithmic growth phase at 25°C and shifted to 37°C at time zero. Samples were taken at intervals, mixed with an equal volume of 2× SDS sample buffer, boiled for 2 min, and analyzed by SDS-PAGE (12.5% polyacrylamide) and immunoblotting with anti-RpoH antiserum essentially as described previously (28). The RpoH band was quantified by densitometry and normalized to the value at 25°C. (B) Log-phase cells grown in synthetic medium at 25°C were shifted to 37°C. Samples taken at the times indicated were pulse-labeled with [³⁵S]methionine for 2 min and treated with trichloroacetic acid, and RpoH was immunoprecipitated, resolved by SDS-PAGE (12.5% polyacrylamide), and quantified as described previously (28). The rate of RpoH synthesis thus obtained was normalized to the value at zero time. (C) Synthesis rate of DnaK was determined by growing and pulse-labeling cells with [³⁵S]methionine as described above for panel B. The cells were treated with trichloroacetic acid and then analyzed by SDS-PAGE (7.5% polyacrylamide) . Radioactivity associated with the DnaK band was determined with a phosphorimager and normalized to the value at zero time.

FIG. 2

Identification of heat-induced rpoH transcripts by S1 mapping. The transcription start site was determined with 10 mg of RNA extracted from non-heat-shocked (−) and heat-shocked (+) cells using rpoH DNA probe fluorescently labeled at the 5′ end. Wild-type ([WT]) cells of A. tumefaciens GV3101 were grown in complete medium at 25°C and heat shocked (shifted to 37°C for 10 min). DNA sequence ladders labeled at the same 5′ end and produced by the Sanger method are shown to the right. The positions of transcription start site and undigested probe are indicated by the closed and open arrows, respectively. The nucleotide sequence of the putative promoter region is shown; the −35 and −10 conserved sequences are underlined, and the start site is circled.

FIG. 3

Heat shock induction of RpoH is abolished by replacing the promoter. (A) Sequence of the rpoH promoter and part of the coding region (underlined) is shown. Lines *1 and *2 represent portions of the sequence that were used or deleted in constructing the transcriptional fusion PrpoH′-lacZ or Plac′-rpoH, respectively (see Materials and Methods). (B) Sequence of a synthetic promoter (Plac′) used to construct Plac′-rpoH, in which rpoH transcription is driven by Plac′. The Rho-independent terminator from E. coli trpA (underlined) (6) was fused to part of the E. coli lac promoter (nucleotides −35 to +1) to prevent readthrough from upstream. (C) A pair of strains with the chromosomal rpoH⁺ gene under the authentic rpoH promoter (KN207) or the Plac′ promoter (KN208) were grown in complete medium to log phase at 25°C and shifted to 37°C. Samples were taken before or after heat shock as indicated, treated, and analyzed by SDS-PAGE (10% polyacrylamide), and RpoH was detected by immunoblotting.

FIG. 4

Transient destabilization of RpoH during the heat shock response. A log-phase culture of wild-type cells (GV3101) grown in synthetic medium at 25°C was divided into two portions, pulse-labeled with [³⁵S]methionine for 2 min, and chased with excess unlabeled methionine. Samples were taken after 3 min and then at 10-min intervals. At the time of second sampling (time zero), one culture was shifted to 37°C (●), whereas the other was kept at 25°C (▪). Immunoprecipitates obtained with anti-RpoH antiserum were analyzed by SDS-PAGE. Averages from three experiments are plotted with standard errors (indicated by the error bars).

FIG. 5

Neither enhanced RpoH level nor de novo protein synthesis is required for induction of heat shock genes. (A) Induction of HSP was analyzed by pulse-labeling cells with [³⁵S]methionine at 25°C or after heat shocking (HS) the cells (shifting the cells to 37°C for 10 min) and resolving the proteins by SDS-PAGE as described in the legend to Fig. 1C. The positions for ClpB, DnaK, and GroEL are indicated on the basis of the immunoblotting data with specific antisera (28). The results with the wild type (KN613), the ΔrpoH mutant (KN201), and derivatives of KN201 carrying intact rpoH (KN207) or Plac′-rpoH (KN208) on the chromosome (indicated by an asterisk) are presented. (B) Induction of dnaK mRNA was analyzed by growing cells as described above for panel A, and chloramphenicol (Cm) was added (+) to a concentration of 100 μg/ml 1 min prior to temperature upshift as indicated. Samples were taken before (−) and 10 min (+) after heat shock (HS), RNA was extracted, and primer extension was performed using a fluorescence-labeled primer for dnaK transcript as described previously (28).

FIG. 6

Effects of changes in the levels of DnaKJ chaperones or GroESL chaperones on the levels of other HSP during steady-state growth. Cells were grown in synthetic medium at 25°C, and proteins were analyzed by SDS-PAGE followed by immunoblotting using specific antisera against E. coli ClpB, DnaK, GroEL, or A. tumefaciens RpoH as indicated to the left. (A) Overproduction of chaperones by multicopy pBBR122 plasmid carrying the dnaKJ or groESL operon in strain KN613. Lanes: 1, pBBR122 (control); 2, pBBR122-dnakJn; 3, pBBR122-dnaKJr; 4, pBBR122-groESLn; 5, pBBR122-groESLr. Both DnaKJ and GroESL were expressed slightly more efficiently when the respective operon was inserted in the plasmid in the direction opposite that of the cat gene (“r” constructs) than when the operon was inserted in the same direction as that of cat (“n” constructs), perhaps due to activity of some uncharacterized promoter(s). (B) Reduced production of DnaK in strains in which the chromosomal dnaK promoter was replaced by the trc promoter in the rpoH⁺ (PrpoH-rpoH) or Plac′-rpoH background. Lanes: 1, KN613 (rpoH⁺ strain [control]); 2, KN614 (dnaKJ driven by the trc promoter in the rpoH⁺ strain); 3, KN209 (Plac′-rpoH strain [control]); 4, KN214 (dnaKJ driven by the trc promoter in the Plac′-rpoH strain). (C) Reduced production of GroESL in which the chromosomal groE promoter was replaced by the trc promoter. Lanes: 1, KN613 (rpoH⁺ strain [control]); 2, KN615 (groESL driven by the trc promoter in the rpoH⁺ strain).

FIG. 7

Time course of change in RpoH activity (LacZ expression from the pUCD-PrpoH′-lacZ reporter plasmid) during the heat shock response. The same set of strains used in Table 4 were grown in synthetic medium at 25°C and shifted to 37°C at time zero. Samples taken at the times indicated were pulse-labeled with [³⁵S]methionine for 2 min, chased with excess unlabeled methionine for 1 min, and treated with 10% trichloroacetic acid in ice. Radiolabeled E. coli ς³²–β-galactosidase fusion protein (GF807-FS [26]) was added as an internal reference to each sample containing the same radioactivity, immunoprecipitated with anti-β-galactosidase antiserum, and resolved by SDS-PAGE (7.5% polyacrylamide). The radioactivities of β-galactosidase bands were normalized to that of the reference protein and are shown as percentages of the maximum synthesis rate in the control (wild-type [WT]) strain. (A) Data for the rpoH⁺ strain (KN613) carrying pBBR122 (control or WT ▪), pBBR122-dnaKJr (Excess DnaKJ ♦), pBBR122-groESLr (Excess GroESL ●), or KN615 (Ptrc-groE) (Reduced GroESL ○) are shown. (B) Data for the pair of Plac′-rpoH strains, KN209 (control or WT ▪) and KN214 (Reduced DnaKJ ⋄), are shown.