Construction and analysis of hybrid Escherichia coli-Bacillus subtilis dnaK genes

Abstract

The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since Bacillus subtilis dnaK was not able to complement an Escherichia coli dnaK null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the B. subtilis DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in E. coli. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in E. coli DnaK affected neither complementation of growth at high temperatures nor propagation of phage lambda but abolished degradation of sigma32.

FIG. 1

Construction of hybrid dnaK genes. The domain structures of the DnaK proteins and the positions of the domain boundaries (E. coli/B. subtilis) are given. Amino acid exchanges caused by the introduction of restriction sites are indicated. By using sequence-specific mutagenesis (14), two different restriction sites were introduced into the two dnaK genes. An AflII site was introduced in the region separating the ATPase and peptide-binding domains, leading to V385L and V356L exchanges in the DnaK proteins of E. coli and B. subtilis, respectively. An SpeI site was created within the interdomain region between the peptide-binding and C-terminal domains of both genes, leading to Q540H and L542V exchanges in the E. coli protein only. The hybrid genes shown here were constructed by using these modified prototype genes.

FIG. 2

Complementation of temperature-sensitive phenotypes and phage λ propagation by plasmid-encoded hybrid dnaK genes. ΔdnaK52, dnaK756 and ΔdnaK52 mutant strains carrying the dnaJ gene under arabinose and IPTG control and containing the different dnaK hybrids were tested for growth at 30 and 40 or 46°C (A) and for propagation of bacteriophage λ at 30°C (B). Growth was assayed by determining the ability of the cells to form colonies or plaques on Luria Bertani agar plates. +, wild-type number and size of colonies or plaques; - -, no colonies or plaques. All strains were grown in the presence of 250 μM IPTG to induce the expression of the dnaK genes, and those carrying pdnaJ also received 0.5% arabinose. Plates were scored for colonies and plaques after 20 h of incubation.

FIG. 3

Amounts of ς³² in the presence of different hybrid proteins. ΔdnaK52 or dnaK756 mutant strains carrying hybrid dnaK genes were grown to mid-exponential phase at 30°C and induced with 250 μM IPTG. Whole-cell fractions corresponding to identical amounts of cell culture were collected before (−) or 2 h after (+) the addition of IPTG, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. The membranes were probed with anti-ς³² antibodies (1:10,000 dilution) and developed by a colorimetric assay as previously described (13).