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. 2004 Feb;5(2):195-200.
doi: 10.1038/sj.embor.7400067. Epub 2004 Jan 9.

In vivo analysis of the overlapping functions of DnaK and trigger factor

Pierre Genevaux  1 , France Keppel, Françoise Schwager, Petra S Langendijk-Genevaux, F Ulrich Hartl, Costa Georgopoulos
Affiliations

In vivo analysis of the overlapping functions of DnaK and trigger factor

Pierre Genevaux et al. EMBO Rep. 2004 Feb.

Abstract

Trigger factor (TF) is a ribosome-bound protein that combines catalysis of peptidyl-prolyl isomerization and chaperone-like activities in Escherichia coli. TF was shown to cooperate with the DnaK (Hsp70) chaperone machinery in the folding of newly synthesized proteins, and the double deletion of the corresponding genes (tig and dnaK) exhibited synthetic lethality. We used a detailed genetic approach to characterize various aspects of this functional cooperation in vivo. Surprisingly, we showed that under specific growth conditions, one can delete both dnaK and tig, indicating that bacterial survival can be maintained in the absence of these two major cytosolic chaperones. The strain lacking both DnaK and TF exhibits a very narrow temperature range of growth and a high level of aggregated proteins when compared to either of the single mutants. We found that, in the absence of DnaK, both the N-terminal ribosome-binding domain and the C-terminal domain of unknown function are essential for TF chaperone activity. In contrast, the central PPIase domain is dispensable. Taken together, our data indicate that under certain conditions, folding of newly synthesized proteins in E. coli is not totally dependent on an interaction with either TF and/or DnaK, and suggest that additional chaperones may be involved in this essential process.

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Figures

Figure 1
Figure 1
TF and DnaK are dispensable for E. coli survival. The genotype of the various bacterial strains is shown at the top. (A) Overnight cultures of wild-type (wt) MC4100 and its various mutant derivatives were serially diluted and spotted on LB agar plates at the indicated temperatures. Immunoblot analysis of endogenous steadystate expression levels of TF, DnaK and GroEL is shown at the bottom of the figure. (B) Aggregated proteins from either wild type or the indicated mutants grown for 4 h at 30°C were separated by 12% SDS–PAGE and stained with Coomassie blue. (C) Effect of various combinations of dnaJ, cbpA and djlA mutations on E. coli growth in the absence of TF, following overnight incubation at the indicated temperature. (+) indicates wild-type gene and (−) indicates null mutant.
Figure 2
Figure 2
TF domain requirements. (A) Complementation of the Ts phenotype of MC4100Δtig ΔdnaKdnaJ on LB ampicillin agar plates by the various TF deletion constructs under the control of the tig native promoter. PPI, PPIase domain; RBD, ribosome-binding domain; UK, TF C-terminal domain of unknown function. (B) Complementation of protein aggregation in the same strain and by the same TF plasmid constructs. A western blot showing the steadystate expression levels of the various TF constructs used is shown at the bottom of the figure. The origin of the constructs is indicated at the top of the figure.
Figure 3
Figure 3
Chaperonins GroES/GroEL partially compensate for the lack of TF and DnaK. Complementation of the Ts phenotype (A) and of protein aggregation (B) of the MC4100Δtig ΔdnaKdnaJ by p29SEN-based IPTG-inducible constructs carrying the genes indicated at the top of the figure. (−) indicates no IPTG, (+) indicates the presence of 1 mM IPTG and (*) indicates aggregated OmpF protein.
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