The Escherichia coli heat shock protein ClpB restores acquired thermotolerance to a cyanobacterial clpB deletion mutant

Abstract

In both prokaryotes and eukaryotes, the heat shock protein ClpB functions as a molecular chaperone and plays a key role in resisting high temperature stress. ClpB is important for the development of thermotolerance in yeast and cyanobacteria but apparently not in Escherichia coli. We undertook a complementation study to investigate whether the ClpB protein from E coli (EcClpB) differs functionally from its cyanobacterial counterpart in the unicellular cyanobacterium Synechococcus sp. PCC 7942. The EcClpB protein is 56% identical to its ClpB1 homologue in Synechococcus. A plasmid construct was prepared containing the entire E coli clpB gene under the control of the Synechococcus clpB1 promoter. This construct was transformed into a Synechococcus clpB1 deletion strain (deltaclpB1) and integrated into a phenotypically neutral site of the chromosome. The full-length EcClpB protein (EcClpB-93) was induced in the transformed Synechococcus strain during heat shock as well as the smaller protein (EcClpB-79) that arises from a second translational start inside the single clpB message. Using cell survival measurements we show that the EcClpB protein can complement the Synechococcus deltaclpB1 mutant and restore its ability to develop thermotolerance. We also demonstrate that both EcClpB-93 and -79 appear to contribute to the degree of acquired thermotolerance restored to the Synechococcus complementation strains.

Fig 1.

Synthesis of E coli ClpB proteins in the Synechococcus ΔclpB1 mutant. (A) Structural representation of the plasmid construct pEcB used to express the E coli clpB gene in the Synechococcus ΔclpB1 strain. The entire E coli clpB gene was cloned into pNSL under the control of the heat shock–inducible Synechococcus clpB1 promoter. The chloramphenicol-resistance gene acting as selectable marker was then inserted upstream of the clpB1 promoter. Flanking this construct were the 2 neutral-site loci (NS 1 and −2) to enable recombination of the resulting plasmid (pEcB) into a phenotypically neutral region of the Synechococcus ΔclpB1 genome (Bustos and Golden 1992). The DNA regions from the dual translational start codons (underlined) for EcClpB-79 and -93 to 13 bases upstream are shown, along with the putative RBS in bold. (B,C) Induction of ClpB proteins in wild-type Synechococcus and the complementation strain EcB. Wild-type (WT) and EcB cultures were shifted from 37 to 48.5°C for 90 minutes. Cellular proteins were isolated at selected times and separated by polyacrylamide gel electrophoresis on the basis of equal Chl content. (B) Immunoblot detection of SyClpC, SyClpB1–93, and –79 in WT, and SyClpC and EcClpB-93 and -79 in EcB using the yeast Hsp104 antibody. (C) Immunoblot detection of EcClpB proteins in EcB using a specific polyclonal antibody. Results are representative of 3 replicates

Fig 2.

Cell survival assay for acquired thermotolerance in EcB strain. Cells of WT, EcB, and ΔclpB1 grown at 37°C were shifted either directly to 54°C for 15 minutes (left column) or first preconditioned at 48.5°C for 1.5 hours before being exposed to 54°C for 15 minutes (right column). At the indicated time points, cells were removed and serially diluted from 1:1 to 1:10 000 and then spotted on BG-11 plates. Shown is a representative thermotolerance experiment of 3 independent replicas

Fig 3.

Quantification of thermotolerance developed in the EcB strain. Synechococcus WT (○), EcB (▪), and ΔclpB1 (▵) cultures grown at 37°C were either directly shifted to 54°C for 15 minutes (A) or first preheated at 48.5°C for 1.5 hours before the shift to 54°C for 15 minutes (B). Average numbers of viable cells after each temperature treatment are expressed as percentages of the value for the 37°C control (100%). All values are averages ± SE for 3 independent experiments

Fig 4.

Increased synthesis of EcClpB-93 in Synechococcus ΔclpB1 mutant. (A) Structural representation of the second construct pEcB1 used to increase the amount of EcClpB-93 protein synthesized in the Synechococcus ΔclpB1 strain. The pEcB1 was made as described for pEcB in Figure 1A, except for the altered RBS (bold) upstream of the ATG start codon (underlined) in base position 1. (B,C) Induction of ClpB proteins in wild-type (WT) Synechococcus and the complementation strains EcB and EcB1. All 3 strains were shifted from 37 to 48.5°C for 90 minutes, and cellular proteins were isolated at selected times and then separated by polyacrylamide gel electrophoresis on the basis of equal Chl content. (B) Immunoblot detection of SyClpC, SyClpB1–93, and –79 in WT, and SyClpC and EcClpB-93 and -79 in EcB1 using the yeast Hsp104 antibody. (C) Immunoblot detection of EcClpB proteins in the EcB and EcB1 strains using a specific polyclonal antibody. Results are representative of 3 replicates

Fig 5.

(A) Cell survival assay for acquired thermotolerance in EcB and EcB1 complementation strains. Thermotolerance experiments and cell survival determinations were done as described in Figure 2. Shown are representative experiments from 3 independent replicas for each strain. (B) Quantification of thermotolerance developed in the EcB and EcB1 strains. Synechococcus complementation strains EcB (▪) and EcB1 (□) grown at 37°C were preheated at 48.5°C for 1.5 hours before the shift to 54°C for 15 minutes. Average numbers of viable cells after each temperature treatment are expressed as percentages of the value for the 37°C control (100%). All values are averages ± SE for 3 independent experiments