A bacteriophage-encoded J-domain protein interacts with the DnaK/Hsp70 chaperone and stabilizes the heat-shock factor σ32 of Escherichia coli.

The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ(32), which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ(32) facilitates RB43 bacteriophage proliferation is discussed.

The Escherichia coli rpoS-dependent htrC gene is not involved in the heat shock response.

We found that a new mutant with a deletion/replacement of the Escherichia coli K-12 htrC gene, a gene previously reported to be required for growth at elevated temperatures, is not temperature sensitive. Furthermore, the original mutants, kindly provided by the original authors, although temperature sensitive, do not have mutations in the open reading frame designated htrC. We found that htrC requires RpoS for enhanced expression in the early stationary phase and is expressed at very low levels until then. The growth of our htrC mutant slowed during the early stationary phase, and the mutant was replaced by its parent in mixed cultures. Since we cannot assign a function or distinctive phenotype to htrC, we suggest that this open reading frame should be given a positional designation, yjaZ, until a specific function is identified.

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

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.

Bacteriophage-encoded cochaperonins can substitute for Escherichia coli's essential GroES protein.

The Escherichia coli chaperonin machine is composed of two members, GroEL and GroES. The GroEL chaperonin can bind 10-15% of E. coli's unfolded proteins in one of its central cavities and help them fold in cooperation with the GroES cochaperonin. Both proteins are absolutely essential for bacterial growth. Several large, lytic bacteriophages, such as T4 and RB49, use the host-encoded GroEL in conjunction with their own bacteriophage-encoded cochaperonin for the correct assembly of their major capsid protein, suggesting a cochaperonin specificity for the in vivo folding of certain substrates. Here, we demonstrate that, when the cochaperonin of either bacteriophage T4 (Gp31) or RB49 (CocO) is expressed in E. coli, the otherwise essential groES gene can be deleted. Thus, it appears that, despite very little sequence identity with groES, the bacteriophage-encoded Gp31 and CocO proteins are capable of replacing GroES in the folding of E. coli's essential, housekeeping proteins.