Trigger Factor can antagonize both SecB and DnaK/DnaJ chaperone functions in Escherichia coli

Abstract

Polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytoplasmic, integral membrane, or exported proteins. In Escherichia coli, the chaperones SecB, Trigger Factor (TF), and DnaK are key players in this process. Here, we report that, as with dnaK or dnaJ mutants, a secB null strain exhibits a strong cold-sensitive (Cs) phenotype. Through suppressor analyses, we found that inactivating mutations in the tig gene encoding TF fully relieve both the Cs phenotype and protein aggregation observed in the absence of SecB. This antagonistic effect of TF depends on its ribosome-binding and chaperone activities but unrelated to its peptidyl-prolyl cis/trans isomerase (PPIase) activity. Furthermore, in contrast to the previously known synergistic action of TF and DnaK/DnaJ above 30 degrees C, a tig null mutation partially suppresses the Cs phenotype exhibited by a compromised DnaK/DnaJ chaperone machine. The antagonistic role of TF is further exemplified by the fact that the secB dnaJ double mutant is viable only in the absence of TF. Finally, we show that, in the absence of TF, more SecA and ribosomes are associated with the inner membrane, suggesting that the presence of TF directly or indirectly interferes with the process of cotranslational protein targeting to the Sec translocon.

Fig. 1.

SecB-dependent phenotypes. (A) The MC4100 ΔsecB strain was transformed with either the p29SEN vector or p29SEN-SecB. Fresh transformants were serially diluted 10-fold and spotted on LB-ampicillin agar plates in the presence of 10 μM IPTG inducer. Plates were incubated for either 18 h at 37 or 46°C or for 6 days at 16°C. (B) Pulse–chase experiments showing the processing of OmpA and maltose-binding protein (MBP) precursors in the MC4100 ΔsecB mutant in the presence of either p29SEN vector or p29SEN-SecB.

Fig. 2.

TF antagonizes SecB. (A) The deletion of the tig gene suppresses the Cs and Ts phenotypes of the ΔsecB mutant. MC4100 and its isogenic mutant derivatives were grown to mid-log phase, serially diluted 10-fold, spotted on LB agar plates, and incubated at the indicated temperatures. (B) Western blot analysis confirming the absence of SecB and/or TF in the mutant strains. (C) Protein aggregation in MC4100 and its isogenic mutant derivatives at the permissive temperature of 37°C. The aggregated proteins were separated by SDS/PAGE and visualized by silver staining.

Fig. 3.

TF domain requirement. (A) Schematic representation of the various p29SEN-based TF constructs used in this work. Light gray, N-terminal domain; white, PPIase domain; black, C domain. (B) Steady-state levels of the TF constructs as judged by Western blot analysis using anti-TF antibodies. (C) Growth of the MC4100 ΔsecB Δtig double mutant transformed with the various p29SEN-based TF constructs. Fresh transformants were serially diluted and spotted on LB-ampicillin agar plates with or without IPTG. (D) Pulse–chase experiments showing the processing of OmpA precursor in the MC4100 ΔsecB Δtig double mutant transformed with the various p29SEN-based TF constructs in the presence of 100 μM IPTG.

Fig. 4.

TF antagonizes the DnaK/DnaJ chaperone machine at low temperatures. (A) Growth of MC4100 and its isogenic Δtig, dnaJ::Tn10-42, and Δtig dnaJ::Tn10-42 mutant derivatives following incubation on LB agar plates at the indicated temperatures. (B) The dnaJ::Tn10-42 mutant allele was introduced by phage P1 transduction into either the ΔsecB or ΔsecB Δtig mutant derivatives in the presence of the various p29SEN-based constructs. −, vector; B, SecB; J, DnaJ. The results of a representative P1 transduction experiment performed at 30°C on LB-tetracycline-ampicillin agar plates supplemented with 10 μM IPTG are shown. (C) Intracellular protein aggregation at 30°C after a 3-h depletion of DnaK/DnaJ in the presence (wt) or in the absence (ΔsecB) of chromosomally encoded SecB. + indicates the presence of 0.5% l-arabinose, and − indicates the absence of l-arabinose and the presence of 0.4% glucose. Western blot analysis of the resulting steady-state levels of DnaK after the 3-h depletion is shown. The nature of the aggregates is shown in SI Table 1 and Fig. 7.

Fig. 5.

TF modulates both SecA and ribosome association with IMs. (A) Western blot analysis of either whole-cell or IM preparations showing SecA, L4, S3, YidC, and FtsA levels from cells grown at 37°C. The MC4100 wild type and its Δtig mutant derivative, as well as the Δtig mutant transformed with either p29SEN vector or p29SEN-TF are shown. Expression of TF from p29SEN-TF was induced with 100 μM IPTG for 1 h. Comparison of cytoplasmic and whole membrane fractions observed under such conditions is shown in SI Fig. 9. (B) Complementation of the Cs phenotype of MC4100 ΔsecB by p29SEN vector or p29SEN-SecA. Fresh transformants were serially diluted 10-fold and spotted on LB-ampicillin agar plates in the presence of 100 μM IPTG and incubated at the indicated temperatures. (C) SecA expression levels in these transformants at 37°C after 2-h induction with 100 μM IPTG. (D) Suppression of the OppA export defect of MC4100 ΔsecB by p29SEN vector or p29SEN-SecA at 23°C in the presence of 100 μM IPTG.